Antigen binding molecules comprising a trimeric tnf family ligand

ABSTRACT

The invention relates to novel TNF family ligand trimer-containing antigen binding molecules comprising (a) at least one moiety capable of specific binding to a target cell antigen, (b) a polypeptide comprising three ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by peptide linkers and (c) a Fc domain composed of a first and a second subunit capable of stable association, and to methods of producing these molecules and to methods of using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to European Patent Application No. EP15161880.8, filed Mar. 31, 2015, the disclosure of which is incorporatedherein by reference in its entirety.

SEQUENCE LISTING

The present application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 29, 2016, isnamed P32693-US_ST25.txt and is 412,194 bytes in size.

FIELD OF THE INVENTION

The invention relates to novel TNF family ligand trimer-containingantigen binding molecules comprising (a) at least one moiety capable ofspecific binding to a target cell antigen, (b) a polypeptide comprisingthree ectodomains of a TNF ligand family member or fragments thereofthat are connected to each other by a peptide linker, and (c) a Fcdomain composed of a first and a second subunit capable of stableassociation. The invention further relates to methods of producing thesemolecules and to methods of using the same.

BACKGROUND

Ligands interacting with molecules of the TNF (tumor necrosis factor)receptor superfamily have pivotal roles in the organization and functionof the immune system. While regulating normal functions such as immuneresponses, hematopoiesis and morphogenesis, the TNF family ligands (alsocalled cytokines) play a role in tumorgenesis, transplant rejection,septic shock, viral replication, bone resorption, rheumatoid arthritisand diabetes (Aggarwal, 2003). Their diverse and important role inimmunity has made them interesting for the development of cancerimmunotherapeutics (Bremer, 2013). The TNF ligand family comprises 18genes encoding 19 type II (i.e. intracellular N terminus andextracellular C-terminus) transmembrane proteins, characterized by thepresence of a conserved C-terminal domain coined the TNF homologydomain' (THD). This domain is responsible for receptor binding and isthus critical for the biological activity of the TNF ligand familymembers. The sequence identity between family members is ˜20-30%(Bodmer, 2002). Members of the TNF ligand family exert their biologicalfunction as self-assembling, noncovalent trimers (Banner et al, 1993).Thus, the TNF family ligands form a trimer that is able to bind to andto activate the corresponding receptors of TNFR superfamily. Somemembers of the TNF ligand family have costimulatory effects on T-cells,meaning that they sustain T-cell responses after initial T cellactivation (Watts, 2005). 4-1BBL, OX40L, GITRL, CD70, CD30L and LIGHTbelong to this group of costimulatory TNF family ligands.

Among several costimulatory molecules, the tumor necrosis factor (TNF)receptor family member OX40 (CD134) plays a key role in the survival andhomeostasis of effector and memory T cells (Croft et al., 2009). OX40(CD134) regulates immune responses against infections, tumors andself-antigens and its expression has been demonstrated on the surface ofT-cells, NKT-cells and NK-cells as well as neutrophils (Baumann et al.,2004) and shown to be strictly inducible or strongly upregulated inresponse to various stimulatory signals. Combined with T-cell receptortriggering, OX40 engagement on T-cells by its natural ligand oragonistic antibodies leads to synergistic activation of the PI3K andNFκB signalling pathways (Song et al., 2008). In turn, this results inenhanced proliferation, increased cytokine receptor and cytokineproduction and better survival of activated T-cells.

4-1BB (CD137), a member of the TNF receptor superfamily, has been firstidentified as a molecule whose expression is induced by T-cellactivation (Kwon and Weissman, 1989). Subsequent studies demonstratedexpression of 4-1BB in T- and B-lymphocytes (Snell et al., 2011; Zhanget al., 2010), NK-cells (Lin et al., 2008), NKT-cells (Kim et al.,2008), monocytes (Kienzle and von Kempis, 2000; Schwarz et al., 1995),neutrophils (Heinisch et al., 2000), mast (Nishimoto et al., 2005) anddendritic cells as well as cells of non-hematopoietic origin such asendothelial and smooth muscle cells (Broll et al., 2001; Olofsson etal., 2008). Expression of 4-1BB in different cell types is mostlyinducible and driven by various stimulatory signals, such as T-cellreceptor (TCR) or B-cell receptor triggering, as well as signalinginduced through co-stimulatory molecules or receptors ofpro-inflammatory cytokines (Diehl et al., 2002; von Kempis et al., 1997;Zhang et al., 2010).

Expression of 4-1BB ligand (4-1BBL or CD137L) is more restricted and isobserved on professional antigen presenting cells (APC) such as B-cells,dendritic cells (DCs) and macrophages. Inducible expression of 4-1BBL ischaracteristic for T-cells, including both αβ and γδ T-cell subsets, andendothelial cells (reviewed in Shao and Schwarz, 2011).

CD137 signaling is known to stimulate IFNy secretion and proliferationof NK cells (Buechele et al., 2012; Lin et al., 2008; Melero et al.,1998) as well as to promote DC activation as indicated by theirincreased survival and capacity to secret cytokines and upregulateco-stimulatory molecules (Choi et al., 2009; Futagawa et al., 2002;Wilcox et al., 2002). However, CD137 is best characterized as aco-stimulatory molecule which modulates TCR-induced activation in boththe CD4+ and CD8+ subsets of T-cells. In combination with TCRtriggering, agonistic 4-1BB-specific antibodies enhance proliferation ofT-cells, stimulate lymphokine secretion and decrease sensitivity ofT-lymphocytes to activation-induced cells death (reviewed in (reviewedin Snell et al., 2011).

In line with these co-stimulatory effects of 4-1BB antibodies on T-cellsin vitro, their administration to tumor bearing mice leads to potentanti-tumor effects in many experimental tumor models (Melero et al.,1997; Narazaki et al., 2010). However, 4-1BB usually exhibits itspotency as an anti-tumor agent only when administered in combinationwith other immunomodulatory compounds (Curran et al., 2011; Guo et al.,2013; Morales-Kastresana et al., 2013; Teng et al., 2009; Wei et al.,2013), chemotherapeutic reagents (Ju et al., 2008; Kim et al., 2009),tumor-specific vaccination (Cuadros et al., 2005; Lee et al., 2011) orradiotherapy (Shi and Siemann, 2006). In vivo depletion experimentsdemonstrated that CD8+ T-cells play the most critical role inanti-tumoral effect of 4-1BB-specific antibodies. However, depending onthe tumor model or combination therapy, which includes anti-4-1BB,contributions of other types of cells such as DCs, NK-cells or CD4+T-cells have been reported (Melero et al., 1997; Murillo et al., 2009;Narazaki et al., 2010; Stagg et al., 2011).

In addition to their direct effects on different lymphocyte subsets,4-1BB agonists can also induce infiltration and retention of activatedT-cells in the tumor through 4-1BB-mediated upregulation ofintercellular adhesion molecule 1 (ICAM1) and vascular cell adhesionmolecule 1 (VCAM1) on tumor vascular endothelium (Palazon et al., 2011).

4-1BB triggering may also reverse the state of T-cell anergy induced byexposure to soluble antigen that may contribute to disruption ofimmunological tolerance in the tumor micro-environment or during chronicinfections (Wilcox et al., 2004).

It appears that the immunomodulatory properties of 4-1BB agonisticantibodies in vivo require the presence of the wild type Fc-portion onthe antibody molecule thereby implicating Fc-receptor binding as animportant event required for the pharmacological activity of suchreagents as has been described for agonistic antibodies specific toother apoptosis-inducing or immunomodulatory members of theTNFR-superfamily (Li and Ravetch, 2011; Teng et al., 2009). However,systemic administration of 4-1BB-specific agonistic antibodies with thefunctionally active Fc domain also induces expansion of CD8+ T-cellsassociated with liver toxicity (Dubrot et al., 2010) that is diminishedor significantly ameliorated in the absence of functional Fc-receptorsin mice. In human clinical trials (ClinicalTrials.gov, NCT00309023),Fc-competent 4-1BB agonistic antibodies (BMS-663513) administered onceevery three weeks for 12 weeks induced stabilization of the disease inpatients with melanoma, ovarian or renal cell carcinoma. However, thesame antibody given in another trial (NCT00612664) caused grade 4hepatitis leading to termination of the trial (Simeone and Ascierto,2012).

Collectively, the available pre-clinical and clinical data clearlydemonstrate that there is a high clinical need for effective 4-1BBagonists. However, new generation drug candidates should not onlyeffectively engage 4-1BB on the surface of hematopoietic and endothelialcells but also be capable of achieving that through mechanisms otherthan binding to Fc-receptors in order to avoid uncontrollable sideeffects. The latter may be accomplished through preferential binding toand oligomerization on tumor-specific or tumor-associated moieties.

Fusion proteins composed of one extracellular domain of a 4-1BB ligandand a single chain antibody fragment (Mueller et al., 2008; Hornig etal., 2012, Kermer et al., 2014) or a single 4-1BB ligand fused to theC-terminus of a heavy chain (Zhang et al, 2007) have been made. EP 1 736482 A1 relates to recombinant 4-1BBL polypeptides comprising the wholeextracellular domain of native 4-1BBL and a cross-linkable peptidedomain. WO 2010/010051 discloses the generation of fusion proteins thatconsist of three TNF ligand ectodomains linked to each other and fusedto an antibody part.

However, there is a need of new antigen binding molecules that combine amoiety capable of preferred binding to tumor-specific ortumor-associated targets with a moiety capable of forming acostimulatory TNF family ligand trimer and that possess sufficientstability to be pharmaceutically useful. The antigen binding moleculesof the present invention comprise both and therefore may be able totrigger TNF receptors not only effectively, but also very selectively atthe desired site thereby reducing side effects.

SUMMARY OF THE INVENTION

This invention provides a TNF family ligand trimer-containing antigenbinding molecule comprising

-   (a) at least one moiety capable of specific binding to a target cell    antigen,-   (b) a polypeptide comprising three ectodomains of a TNF ligand    family member or fragments thereof that are connected to each other,    optionally by peptide linkers, and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association.

In one aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising

-   (a) at least one moiety capable of specific binding to a target cell    antigen,-   (b) a polypeptide comprising three ectodomains of a TNF ligand    family member or fragments thereof that are connected to each other    by peptide linkers and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association.

In another aspect, the TNF family ligand trimer containing antigenbinding molecule comprises

-   (a) at least one moiety capable of specific binding to a target cell    antigen,-   (b) a polypeptide comprising three ectodomains of a TNF ligand    family member or fragments thereof that are connected to each other    by peptide linkers and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association, wherein the polypeptide comprising the three    ectodomains of a TNF ligand family member or fragments thereof that    are connected to each other by peptide linkers is fused to the N- or    C-terminal amino acid of one of the two subunits of the Fc domain,    optionally through a peptide linker.

In a particular aspect, the TNF family ligand trimer-containing antigenbinding molecule comprises (a) at least one moiety capable of specificbinding to a target cell antigen, (b) a polypeptide comprising threeectodomains of a TNF ligand family member or fragments thereof that areconnected to each other by peptide linkers and (c) a Fc domain composedof a first and a second subunit capable of stable association, whereinthe TNF ligand family member costimulates human T-cell activation. Thus,the TNF ligand family member is a costimulatory TNF family ligand. Inparticular, the costimulatory TNF family ligand is selected from thegroup consisting of 4-1BBL, OX40L, GITRL, CD70, CD30L and LIGHT, moreparticularly the costimulatory TNF family ligand is selected from 4-1BBLand OX40L.

In one aspect, the TNF ligand family member is 4-1BBL.

In a further aspect, the ectodomain of a TNF ligand family membercomprises the amino acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:99, SEQID NO: 100, SEQ ID NO:101 and SEQ ID NO:102, particularly the amino acidsequence of SEQ ID NO:1 or SEQ ID NO:99.

In a particular aspect, the ectodomain of a TNF ligand family member orfragment thereof comprises the amino acid sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ IDNO:4, particularly the amino acid sequence of SEQ ID NO:1.

In a further aspect, the TNF family ligand trimer-containing antigenbinding molecule of the invention comprises

-   (a) at least one moiety capable of specific binding to a target cell    antigen and-   (b) a polypeptide comprising the amino acid sequence of SEQ ID NO:5    or SEQ ID NO:103, and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association. More particularly, the polypeptide comprises the    amino acid sequence of SEQ ID NO:5.

In another aspect, the TNF ligand family member is OX40L.

In a particular aspect, the ectodomain of a TNF ligand family membercomprises the amino acid sequence selected from the group consisting ofSEQ ID NO:6 and SEQ ID NO:7, particularly the amino acid sequence of SEQID NO:6.

In a further aspect, the TNF family ligand trimer-containing antigenbinding molecule of of the invention comprises

-   (a) at least one moiety capable of specific binding to a target cell    antigen and-   (b) a polypeptide comprising the amino acid sequence of SEQ ID NO:8,    and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association.

In one aspect, the invention relates to the TNF family ligandtrimer-containing antigen binding molecule as described herein before,wherein the polypeptide comprising three ectodomains of a TNF ligandfamily member or fragments thereof is fused at the C-terminal amino acidto the N-terminal amino acid of one of the subunits of the Fc domain. Inparticular, provided is a TNF family ligand trimer-containing antigenbinding molecule comprising

-   (a) at least one moiety capable of specific binding to a target cell    antigen,-   (b) a polypeptide comprising three ectodomains of a TNF ligand    family member or fragments thereof that are connected to each other    by a peptide linkers and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association, wherein the polypeptide comprising three    ectodomains of a TNF ligand family member or fragments thereof is    fused at the C-terminal amino acid to the N-terminal amino acid of a    CH2 domain in the Fc domain.

In another aspect, In another aspect, the TNF family ligandtrimer-containing antigen binding molecule is one, wherein thepolypeptide comprising three ectodomains of a TNF ligand family memberor fragments thereof is fused at the N-terminal amino acid to aC-terminal amino acid of one of the subunits of the Fc domain, e.g. thepolypeptide comprising three ectodomains of a TNF ligand family memberor fragments thereof is fused at the N-terminal amino acid to theC-terminal amino acid of a CH3 domain in the Fc domain.

In another aspect, the invention relates to a TNF family ligandtrimer-containing antigen binding molecule comprising

-   (a) at least one moiety capable of specific binding to a target cell    antigen,-   (b) a polypeptide comprising three ectodomains of a TNF ligand    family member or fragments thereof that are connected to each other    by peptide linkers and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association, wherein the moiety capable of specific binding    to a target cell antigen is not fused to the polypeptide comprising    three ectodomains of a TNF ligand family member or fragments    thereof.

In a further aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule as defined herein before,wherein the moiety capable of specific binding to a target cell antigenis selected from the group consisting of an antibody, an antibodyfragment and a scaffold antigen binding protein.

In one aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule as defined herein before,wherein the moiety capable of specific binding to a target cell antigenis an antibody.

In another aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule as defined herein before,wherein the moiety capable of specific binding to a target cell antigenis an antibody fragment. In particular, the antibody fragment isselected from the group consisting of a Fab molecule, a crossover Fabmolecule, a single chain Fab molecule, a Fv molecule, a scFv molecule, asingle domain antibody, and aVH.

In a further aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule as defined herein before,wherein the moiety capable of specific binding to a target cell antigenis a scaffold antigen binding protein.

Thus, in a further aspect, the invention relates to a TNF family ligandtrimer-containing antigen binding molecule as defined herein before,wherein the moiety capable of specific binding to a target cell antigenis selected from the group consisting of an antibody fragment, a Fabmolecule, a crossover Fab molecule, a single chain Fab molecule, a Fvmolecule, a scFv molecule, a single domain antibody, an aVH and ascaffold antigen binding protein.

In a particular aspect, the invention is concerned with a TNF familyligand trimer-containing antigen binding molecule as defined above,wherein the moiety capable of specific binding to a target cell antigenis a Fab molecule capable of specific binding to a target cell antigen.

In another aspect, provided is a TNF family ligand trimer-containingantigen binding molecule of the invention, wherein the target cellantigen is selected from the group consisting of Fibroblast ActivationProtein (FAP), Carcinoembryonic Antigen (CEA), Melanoma-associatedChondroitin Sulfate Proteoglycan (MCSP), Epidermal Growth FactorReceptor (EGFR), CD19, CD20 and CD33.

In a particular aspect, the target cell antigen is Fibroblast ActivationProtein (FAP). In one aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule as decribed herein before,wherein the moiety capable of specific binding to FAP comprises (a) a VHdomain comprising (i) CDR-H1 comprising the amino acid sequence of SEQID NO:9, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:10and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:11, anda VL domain comprising (iv) CDR-L1 comprising the amino acid sequence ofSEQ ID NO:12, (v) CDR-L2 comprising the amino acid sequence of SEQ IDNO:13 and (vi) CDR-L3 comprising the amino acid sequence of SEQ IDNO:14, or

-   (b) a VH domain comprising (i) CDR-H1 comprising the amino acid    sequence of SEQ ID NO:104, (ii) CDR-H2 comprising the amino acid    sequence of SEQ ID NO:105 and (iii) CDR-H3 comprising the amino acid    sequence of SEQ ID NO:106, and a VL domain comprising (iv) CDR-L1    comprising the amino acid sequence of SEQ ID NO:107, (v) CDR-L2    comprising the amino acid sequence of SEQ ID NO:108 and (vi) CDR-L3    comprising the amino acid sequence of SEQ ID NO:109.

In one aspect, the moiety capable of specific binding to FAP comprises aVH domain comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:9, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:10 and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:11, and a VL domain comprising (iv) CDR-L1 comprising the amino acidsequence of SEQ ID NO:12, (v) CDR-L2 comprising the amino acid sequenceof SEQ ID NO:13 and (vi) CDR-L3 comprising the amino acid sequence ofSEQ ID NO:14. In another particular aspect, the moiety capable ofspecific binding to FAP comprises a VH domain comprising (i) CDR-H1comprising the amino acid sequence of SEQ ID NO:104, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:105 and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:106, and a VL domaincomprising (iv) CDR-L1 comprising the amino acid sequence of SEQ IDNO:107, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:108and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:109.

In a further aspect, the moiety capable of specific binding to FAPcomprises a variable heavy chain comprising an amino acid sequence ofSEQ ID NO:16 and a variable light chain comprising an amino acidsequence of SEQ ID NO:17 or wherein the moiety capable of specificbinding to FAP comprises a variable heavy chain comprising an amino acidsequence of SEQ ID NO:110 and a variable light chain comprising an aminoacid sequence of SEQ ID NO:111.

In another aspect, the target cell antigen is CEA. In one aspect, theinvention provides a TNF family ligand trimer-containing antigen bindingmolecule as decribed herein before, wherein the moiety capable ofspecific binding to CEA comprises a VH domain comprising (i) CDR-H1comprising the amino acid sequence of SEQ ID NO:112, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:113 and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:114, and a VL domaincomprising (iv) CDR-L1 comprising the amino acid sequence of SEQ IDNO:115, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:116and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:117. Inparticular, the moiety capable of specific binding to CEA comprises avariable heavy chain comprising an amino acid sequence of SEQ ID NO:118and a variable light chain comprising an amino acid sequence of SEQ IDNO:119.

In yet another aspect, the target cell antigen is CD19. In one aspect,the invention provides a TNF family ligand trimer-containing antigenbinding molecule as decribed herein before, wherein the moiety capableof specific binding to CD19 comprises (a) a VH domain comprising (i)CDR-H1 comprising the amino acid sequence of SEQ ID NO:120, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:121 and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:122, and a VL domaincomprising (iv) CDR-L1 comprising the amino acid sequence of SEQ IDNO:123, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:124and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:125, or(b) a VH domain comprising (i) CDR-H1 comprising the amino acid sequenceof SEQ ID NO:128, (ii) CDR-H2 comprising the amino acid sequence of SEQID NO:129 and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:130, and a VL domain comprising (iv) CDR-L1 comprising the amino acidsequence of SEQ ID NO:131, (v) CDR-L2 comprising the amino acid sequenceof SEQ ID NO:132 and (vi) CDR-L3 comprising the amino acid sequence ofSEQ ID NO:133. In particular, the the moiety capable of specific bindingto CD19 comprises a variable heavy chain comprising an amino acidsequence of SEQ ID NO:126 and a variable light chain comprising an aminoacid sequence of SEQ ID NO:127 or wherein the moiety capable of specificbinding to FAP comprises a variable heavy chain comprising an amino acidsequence of SEQ ID NO:134 and a variable light chain comprising an aminoacid sequence of SEQ ID NO:135.

In a further aspect, provided is a TNF family ligand trimer-containingantigen binding molecule as described herein before, wherein the Fcdomain composed of a first and a second subunit capable of stableassociation is an IgG, particularly an IgG1 Fc domain or an IgG4 Fcdomain. In particular, the Fc domain composed of a first and a secondsubunit capable of stable association is an IgG1 Fc domain.

In another aspect, the invention is concerned with a TNF family ligandtrimer-containing antigen binding molecule as defined herein before,comprising (c) an Fc domain composed of a first and a second subunitcapable of stable association, wherein the Fc domain comprises one ormore amino acid substitution that reduces binding to an Fc receptor, inparticular towards Fcy receptor.

In particular, the Fc domain comprises amino acid substitutions atpositions 234 and 235 (EU numbering) and/or 329 (EU numbering). Moreparticularly, provided is a trimeric TNF family ligand-containingantigen binding molecule according to the invention which comprises anFc domain with the amino acid substitutions L234A, L235A and P329G (EUnumbering) in the IgG heavy chains.

In a particular aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule as described herein before,wherein the Fc domain is an IgG1 Fc domain and comprises amino acidsubstitutions at positions 234 and 235 (EU numbering) and 329 (EUnumbering) of the IgG heavy chains.

In another aspect, provided is a monovalent TNF family ligandtrimer-containing antigen binding molecule as described herein before,comprising one moiety capable of specific binding to a target cellantigen.

In particular, the TNF family ligand trimer-containing antigen bindingmolecule of the invention comprising (c) an Fc domain composed of afirst and a second subunit capable of stable association furthercomprises (a) a Fab molecule capable of specific binding to a targetcell antigen, wherein the Fab heavy chain is fused at the C-terminus tothe N-terminus of a CH2 domain in the Fc domain.

Furthermore, the invention provides a TNF family ligandtrimer-containing antigen binding molecule as described herein before,wherein the antigen binding molecule comprises

-   (a) a heavy chain and a light chain, both comprising a Fab molecule    capable of specific binding to a target cell antigen, and-   (b) a fusion protein comprising the amino acid sequence of SEQ ID    NO:15.

In another aspect, provided is a TNF family ligand trimer-containingantigen binding molecule as described hereinbefore, wherein the antigenbinding molecule comprises

-   (a) a heavy chain and a light chain, both comprising a Fab molecule    capable of specific binding to a target cell antigen, and-   (b) the fusion protein comprising the amino acid sequence of SEQ ID    NO:20.

In a further aspect, the TNF family ligand trimer-containing antigenbinding molecule comprises

-   (i) a first heavy chain comprising the VH domain of a Fab molecule    capable of specific binding to a target cell antigen,-   (ii) a light chain comprising the VL domain of a Fab molecule    capable of specific binding to a target cell antigen, and-   (iii) a second heavy chain comprising the amino acid sequence of SEQ    ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8.

In a particular aspect, the TNF family ligand trimer-containing antigenbinding molecule comprises (i) a first heavy chain comprising a VHdomain comprising an amino acid sequence of SEQ ID NO:16 or a VH domaincomprising an amino acid sequence of SEQ ID NO:110, (ii) a light chaincomprising a VL domain comprising an amino acid sequence of SEQ ID NO:17or a VL domain comprising an amino acid sequence of SEQ ID NO:111, and(iii) a second heavy chain comprising the amino acid sequence of SEQ IDNO:5 or SEQ ID NO:103 or SEQ ID NO:8. In another aspect, TNF familyligand trimer-containing antigen binding molecule comprises (i) a firstheavy chain comprising a VH domain comprising an amino acid sequence ofSEQ ID NO:118, (ii) a light chain comprising a VL domain comprising anamino acid sequence of SEQ ID NO:119, and (iii) a second heavy chaincomprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:103 orSEQ ID NO:8. In a further aspect, the TNF family ligandtrimer-containing antigen binding molecule comprises (i) a first heavychain comprising a VH domain comprising an amino acid sequence of SEQ IDNO:126 or a VH domain comprising an amino acid sequence of SEQ IDNO:134, (ii) a light chain comprising a VL domain comprising an aminoacid sequence of SEQ ID NO:127 or a VL domain comprising an amino acidsequence of SEQ ID NO:135, and (iii) a second heavy chain comprising theamino acid sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8.

In another aspect, provided is a bivalent TNF family ligandtrimer-containing antigen binding molecule, comprising two moietiescapable of specific binding to a target cell antigen.

In one aspect, the TNF family ligand trimer-containing antigen bindingmolecule comprises

-   (i) a first heavy chain comprising a VH domain of a Fab molecule    capable of specific binding to a target cell antigen,-   (ii) two light chains comprising each the VL domain of a Fab    molecule capable of specific binding to a target cell antigen, and-   (iii) a second heavy chain comprising a VH domain of a Fab molecule    capable of specific binding to a target cell antigen and the amino    acid sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8.

In a particular aspect, the TNF family ligand trimer-containing antigenbinding molecule comprises (i) a first heavy chain comprising a VHdomain comprising an amino acid sequence of SEQ ID NO:16 or a VH domaincomprising an amino acid sequence of SEQ ID NO:110, (ii) two lightchains comprising each a VL domain comprising an amino acid sequence ofSEQ ID NO:17 or a VL domain comprising an amino acid sequence of SEQ IDNO:111, and (iii) a second heavy chain comprising a VH domain comprisingan amino acid sequence of SEQ ID NO:16 or a VH domain comprising anamino acid sequence of SEQ ID NO:110 and the amino acid sequence of SEQID NO:5 or SEQ ID NO:103 or SEQ ID NO:8. In another aspect, the TNFfamily ligand trimer-containing antigen binding molecule comprises (i) afirst heavy chain comprising a VH domain comprising an amino acidsequence of SEQ ID NO:118, (ii) two light chains comprising each a VLdomain comprising an amino acid sequence of SEQ ID NO:119, and (iii) asecond heavy chain comprising a VH domain comprising an amino acidsequence of SEQ ID NO:118 and the amino acid sequence of SEQ ID NO:5 orSEQ ID NO:103 or SEQ ID NO:8. In yet another aspect, the TNF familyligand trimer-containing antigen binding molecule comprises (i) a firstheavy chain comprising a VH domain comprising an amino acid sequence ofSEQ ID NO:126 or a VH domain comprising an amino acid sequence of SEQ IDNO:134, (ii) two light chains comprising a VL domain comprising an aminoacid sequence of SEQ ID NO:127 or a VL domain comprising an amino acidsequence of SEQ ID NO:135, and (iii) a second heavy chain comprising aVH domain comprising an amino acid sequence of SEQ ID NO:126 or a VHdomain comprising an amino acid sequence of SEQ ID NO:134 and the aminoacid sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8.

In a particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as described herein before,wherein the antigen binding molecule comprises

-   (i) a variable heavy chain comprising the amino acid sequence of SEQ    ID NO:16,-   (ii) a variable light chain comprising the amino acid sequence of    SEQ ID NO:17, and-   (iii) a polypeptide comprising the amino acid sequence of SEQ ID    NO:5.

More particularly, provided is a TNF family ligand trimer-containingantigen binding molecule as described herein before, wherein the antigenbinding molecule comprises

-   (i) a heavy chain comprising the amino acid sequence of SEQ ID    NO:18,-   (ii) a light chain comprising the amino acid sequence of SEQ ID    NO:19, and-   (iii) a fusion protein comprising the amino acid sequence of SEQ ID    NO:15.

In a particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as described herein before,wherein the antigen binding molecule comprises

-   (i) a variable heavy chain comprising the amino acid sequence of SEQ    ID NO:16,-   (ii) a variable light chain comprising the amino acid sequence of    SEQ ID NO:17, and-   (iii) a polypeptide comprising the amino acid sequence of SEQ ID    NO:8.

More particularly, provided is a TNF family ligand trimer-containingantigen binding molecule as described herein before, wherein the antigenbinding molecule comprises

-   (i) a heavy chain comprising the amino acid sequence of SEQ ID    NO:18,-   (ii) a light chain comprising the amino acid sequence of SEQ ID    NO:19, and-   (iii) a fusion protein comprising the amino acid sequence of SEQ ID    NO:20.

According to another aspect of the invention, there is provided anisolated polynucleotide encoding a TNF family ligand trimer-containingantigen binding molecule as defined herein before. The invention furtherprovides a vector, particularly an expression vector, comprising theisolated polynucleotide of the invention and a host cell comprising theisolated polynucleotide or the vector of the invention. In someembodiments the host cell is a eukaryotic cell, particularly a mammaliancell.

In another aspect, provided is a method for producing the TNF familyligand trimer-containing antigen binding molecule of the invention,comprising the steps of (i) culturing the host cell of the inventionunder conditions suitable for expression of the antigen bindingmolecule, and (ii) recovering the antigen binding molecule. Theinvention also encompasses a TNF family ligand trimer-containing antigenbinding molecule produced by the method of the invention.

The invention further provides a pharmaceutical composition comprisingthe TNF family ligand trimer-containing antigen binding molecule of theinvention and at least one pharmaceutically acceptable excipient.

Also encompassed by the invention is the TNF family ligandtrimer-containing antigen binding molecule of the invention, or thepharmaceutical composition of the invention, for use as a medicament. Inone aspect, provided is a TNF family ligand trimer-containing antigenbinding molecule of the invention, or the pharmaceutical composition ofthe invention, for use in the treatment of a disease in an individual inneed thereof. In a specific embodiment, provided is the TNF familyligand trimer-containing antigen binding molecule of the invention, orthe pharmaceutical composition of the invention, for use in thetreatment of cancer.

Also provided is the use of the TNF family ligand trimer-containingantigen binding molecule of the invention for the manufacture of amedicament for the treatment of a disease in an individual in needthereof, in particular for the manufacture of a medicament for thetreatment of cancer, as well as a method of treating a disease in anindividual, comprising administering to said individual atherapeutically effective amount of a composition comprising the TNFfamily ligand trimer-containing antigen binding molecule of theinvention in a pharmaceutically acceptable form. In a specificembodiment, the disease is cancer. In any of the above embodiments theindividual is preferably a mammal, particularly a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show polypeptides comprising three ectodomains of a TNFligand family member or fragments thereof that are connected to eachother by peptide linkers. FIG. 1A shows a polypeptide comprising threeectodomains of 4-1BBL that are connected to each other by peptidelinkers and FIG. 1B shows a polypeptide comprising three ectodomains ofOX40L that are connected to each other by peptide linkers.

FIGS. 2A and 2B schematically show variants (2A) and (2B) of themonovalent TNF ligand trimer-containing antigen binding molecules of theinvention. The three TNF ligands are symbolized by the grey areas withwhite points. The white areas with black points symbolize the Fc part,the thick black point stands for the knob-into-hole modification.

A representative example of the TNF ligand trimer-containing antigenbinding molecule of the invention is a FAP-targeted trimeric 4-1BBligand Fc (kih) P329G LALA fusion antigen binding molecule as shown inFIG. 3A. Its preparation is described in Examples 1.1 and 1.3. FIG. 3Bshows the “untargeted” variant (control 1) comprising a DP47 Fabmolecule and the single chain trimeric 4-1BB ligand Fc (kih) P329G LALAfusion antigen binding molecule as prepared in Example 2. FIG. 3C is adrawing of the monomeric 4-1BB Fc(kih) construct as prepared in Example4.

Another example of the TNF ligand trimer-containing antigen bindingmolecule of the invention is a FAP-targeted trimeric OX40 ligand Fc(kih) P329G LALA fusion antigen binding molecule as shown in FIG. 4A.Its preparation is described in Example 6. FIG. 4B shows the“untargeted” variant comprising a DP47 Fab molecule and the single chaintrimeric OX40 ligand Fc (kih) P329G LALA fusion molecule as prepared inExample 7. FIG. 4C is a drawing of the monomeric OX40 Fc(kih) constructas prepared in Example 8.

FIGS. 5A-D show the binding of FAP-targeted 4-1BB ligandtrimer-containing Fc (kih) fusion molecule (compound S1) to recombinant4-1BB Fc (kih) receptor as assessed by surface plasmon resonance. InFIG. 5A the setup of the assay is shown, 4-1BB Fc (kih) is immobilizedon a SA chip. FIG. 5B shows binding to human 4-1BB Fc (kih), FIG. 5Cbinding to cynomolgus 4-1BB Fc (kih) and FIG. 5D binding to murine 4-1BBFc (kih).

The binding of DP47 containing 4-1BB ligand trimer-containing Fc (kih)fusion antigen binding molecule (Control 1) to recombinant 4-1BB Fc kihreceptor is shown in FIGS. 6A-C. 4-1BB Fc (kih) is immobilized on a SAchip. FIG. 6A shows binding to human 4-1BB Fc (kih), FIG. 6B binding tocynomolgus 4-1BB Fc (kih) and FIG. 6C binding to murine 4-1BB Fc (kih).

FIGS. 7A-E illustrate the binding of recombinant human 4-1BB toFAP-targeted 4-1BB ligand trimer-containing Fc (kih) fusion antigenbinding molecule (compound Si). In FIG. 7A the setup of the affinitymeasurement is shown. FIGS. 7B and 7C show the binding of human 4-1BBavi-His tag to the FAP-targeted 4-1BB ligand trimer-containing Fc (kih)fusion antigen binding molecule (FAP-targeted single chain trimeric4-1BBL) and FIGS. 7D and 7E show the binding of human 4-1BB avi-His tagto DP47 containing 4-1BB ligand trimer-containing Fc (kih) fusionantigen binding molecule (DP47-containing single chain trimeric 4-1BBLmolecule).

FIGS. 8A-D shows the binding of FAP-targeted OX40 ligandtrimer-containing Fc (kih) fusion antigen binding molecule (compoundS10) to recombinant OX40 Fc (kih) receptor as assessed by surfaceplasmon resonance. In FIG. 8A the setup of the assay is shown, OX40 Fc(kih) is immobilized on a SA chip. FIG. 8B shows binding to human OX40Fc (kih), FIG. 8C binding to cynomolgus OX40 Fc (kih) and FIG. 8Dbinding to murine OX40 Fc (kih).

In FIGS. 9A-C, the binding of DP47-containing OX40 ligandtrimer-containing Fc (kih) fusion molecule (Control 3) to recombinantOX40 Fc kih receptor is demonstrated. OX40 Fc (kih) is immobilized on aSA chip. FIG. 9A shows binding to human OX40 Fc (kih), FIG. 9B bindingto cynomolgus OX40 Fc (kih) and FIG. 9C binding to murine OX40 Fc (kih).

FIGS. 10A-C show the binding of recombinant human OX40 to FAP-targetedOX40 ligand trimer-containing Fc (kih) fusion antigen binding molecule(Compound S10). In FIG. 10A the setup of the affinity measurement isshown. In FIG. 10B the binding of human OX40 avi-His tag to theFAP-targeted OX40 ligand trimer-containing Fc (kih) fusion antigenbinding molecule (FAP-targeted single chain OX40 ligand) is shown andFIG. 10C illustrates the binding of human 4-1BB avi-His tag toDP47-containing OX40 ligand trimer-containing Fc (kih) fusion molecule(DP47-containing single chain trimeric OX40L).

4-1BB and OX40 are not expressed on resting (naïve) human PBMCs (FIGS.11A, 11C, 11E and 11G, FIGS. 12A and 12C). After activation of humanPBMCs 4-1BB and OX40 are up-regulated on CD4⁺ and CD8⁺ T cells whereashuman CD8⁺ T cells (FIGS. 11B, 11F, 11K and 11M and 12B) show a 10 timeshigher 4-1BB but a 4 times lower OX40 upregulation than on CD4+ T cells(FIGS. 11D, 11H, 11J and 11L and 12D). As shown, FAP-targeted singlechain trimeric 4-1BBL or OX40L Fc(kih) fusion antigen binding molecules(filled circle) and DP47-containing (“untargeted”) single chain trimeric4-1BBL and OX40L (open circle, dotted line) show a similar binding toactivated CD4⁺ and CD8⁺ T cells but no binding to naïve human T cells.Therefore independent of FAP- or DP47-binding, both single chaintrimeric 4-1BBL and single chain trimeric OX40L show similar binding tohuman 4-1BB or OX40 expressing cells. In the absence of human 4-1BB orOX40 expressing cells no binding can be observed (resting, naïve cells).Shown is the binding as median of fluorescence intensity (MFI) of redmacrophytic algae Phycoerythrin (R-PE)- or Fluorescein isothiocyanate(FITC)-conjugated anti-human IgG Fcγ-specific goat IgG F(ab′)₂ fragmentwhich is used as secondary detection antibody. MFI was measured by flowcytometry and baseline corrected by subtracting the MFI of the blankcontrol. The x-axis shows the concentration of FAP- or DP47-containingsingle chain trimeric 4-1BBL or OX40L Fc(kih) fusion molecules.

FIGS. 11A-D relate to the binding of FAP-targeted 4-1BB ligandtrimer-containing Fc(kih) fusion antigen binding molecule (Compound S1,filled circles) or DP47-containing 4-1BB ligand trimer-containingFc(kih) fusion molecule (Control 1, open circles) to resting (naive) oractivated human PMBCs. Specifically, the binding to resting (naive) oractivated human CD8⁺ T cells is shown in the upper two graphs and toresting (naive) or activated human CD4⁺ T cells in the two graphs below,respectively.

FIGS. 11E-H show the binding of monovalent targeted single chaintrimeric 4-1BB ligand Fc (kih) fusion antigen binding molecules toresting and activated human T cells. 4-1BB is not expressed on resting(naive) CD4 or CD8 T cells isolated from the blood of healthy donors.After activation 4-1BB is upregulated on CD4⁺ (see graph right below)and CD8⁺ (see graph right above) T cells whereas CD8⁺ T cells show a5-10 times higher 4-1BB expression than CD4⁺ T cells (depending on thedonor). As shown, all monovalent targeted single chain trimeric 4-1BBLFc (kih) fusion molecules bound similar to 4-1BB⁺ human T cells,independent of their targeting. Shown is on the the y-axis the mean offluorescence intensity (MFI) of the R-PE-conjugated anti-human IgG Fcγfragment-specific F(ab′)₂ goat IgG fragment. On the x-axis the testedconcentration of the different targeted single chain trimeric 4-1BBligand Fc (kih) fusion antigen binding molecules (compounds S1, S3 andS5) are indicated. Values were baseline corrected by subtracting the MFIof blank control.

In FIGS. 11J-M it is shown the binding of monovalent versus bivalenttargeted single chain trimeric 4-1BB ligand Fc (kih) fusion antigenbinding molecules to activated human T cells. After activation 4-1BB isupregulated on CD4⁺ (left hand side) and CD8+ (right hand side) T cells.As shown, all monovalent and bivalent targeted single chain trimeric4-1BBL Fc (kih) fusion antigen binding molecules bound similar to 4-1BB⁺human T cells, independent of their targeting. Shown is on the they-axis the mean of fluorescence intensity (MFI) of the R-PE-conjugatedanti-human IgG Fcγ fragment-specific F(ab′)2 goat IgG fragment. On thex-axis the tested concentration of the different targeted single chaintrimeric 4-1BB ligand Fc (kih) fusion antigen binding molecules(Compounds S1, S3, S2 and S4) are indicated. Values were baselinecorrected by subtracting the MFI of blank control.

FIGS. 12A-D show the binding of FAP-targeted OX40 ligandtrimer-containing Fc(kih) fusion antigen binding molecule (Compound S10,filled circles) or DP47-containing OX40 ligand trimer-containing Fc(kih)fusion molecule (DP47 single chain OX40L trimer, Control 3, opencircles) to resting (naive) or activated human PMBCs. Specifically, thebinding to resting (naive) or activated human CD8⁺ T cells is shown inFIGS. 12A and 12B, respectively, and to resting (naive) or activatedhuman CD4⁺ T cells in FIG. 12C and 12D, respectively. The binding ofDP47 human IgG1 P329G LALA is symbolized by the filled diamonds(negative control).

FIGS. 13A-E shows the binding of single chain TNF ligandtrimer-containing Fc(kih) fusion antigen binding molecules (compound S1and S10, respectively) to transgenic modified mouse embryonic fibroblastNIH/3T3-huFAP clone 39 (FIG. 13A) or to fibroblast activation protein(FAP)-expressing human WM266-4 cell line (FIG. 13B). These cells expresshigh levels of human fibroblast activation protein (huFAP). Shown is thebinding as median of fluorescence intensity (MFI) of Fluoresceinisothiocyanate (FITC)-labeled anti-human IgG Fcγ-specific goat IgGF(ab′)2 fragment which is used as secondary detection antibody. MFI wasmeasured by flow cytometry. The x-axis shows the concentration ofFAP-targeted or DP47-targeted (“untargeted”) single chain trimeric4-1BBL or OX40L antigen binding molecule. In FIG. 13A it is shown thatonly the FAP-targeted single chain trimeric 4-1BBL molecule (CompoundS1, filled circles), but not the DP47-containing single chain trimeric4-1BBL antigen binding molecule (open circles, dotted line) binds toNIH/3T3-huFAP clone 39 cells, FIG. 13B demonstrates that only theFAP-targeted single chain trimeric OX40L antigen binding molecule(Compound S10, filled circles), but not the DP47-containing single chaintrimeric OX40 antigen binding molecule (open circles, dotted line) bindsto WM266-4 cells. As a negative control DP47 human IgG1 P329G LALAsymbolized by the filled diamonds did also not bind.

In FIGS. 13C and 13D it is shown the binding of monovalent versusbivalent FAP (4B9 or 28H1)-targeted single chain trimeric 4-1BB ligandFc (kih) fusion antigen binding molecules to FAP-expressing tumor cells.As shown, the FAP (4B9)-targeted molecules show the strongest binding toFAP with a lower EC₅₀ value. With the strong FAP (4B9) binder we seeonly very small differences in MFI or EC₅₀ between the monovalent andthe bivalent targeted single chain trimeric 4-1BB ligand (kih) fusionantigen molecules (compounds S3 and S2, respectively). This is differentfor the weaker FAP-binder 28H1 (compound S1). Here a bivalent binding(shown with the bivalent binding FAP (28H1)-targeted huIgG1 P329G LALA)gives a lower MFI. DP47-untargeted molecules do not bind to the FAP+tumor cells and are therefore truly untargeted. Shown is on the they-axis the mean of fluorescence intensity (MFI) of the R-PE-conjugatedanti-human IgG Fcγ fragment-specific F(ab')2 goat IgG fragment. On thex-axis the tested concentration of the different targeted single chaintrimeric 4-1BB ligand Fc (kih) fusion antigen binding molecules areindicated.

FIG. 13E shows the binding of monovalent versus bivalent CEA-targetedsingle chain trimeric 4-1BB ligand Fc (kih) fusion antigen bindingmolecules to a CEA-expressing tumor cell line. The binding of thebivalent CEA-targeted single chain trimeric 4-1BB ligand Fc (kih) fusionantigen binding molecule (compound S4) gives a lower MFI than that ofthe monovalent CEA-targeted single chain trimeric 4-1BB ligand Fc (kih)fusion antigen binding molecule (compound S5). This can be explained bythe occupation of two CEA-molecules on the surface of the tumor cell atthe same time. The DP47-untargeted molecules (Control 1 and 2,respectively) do not bind to the CEA+MKN45 tumor cells and are thereforetruly untargeted. Shown is on the the y-axis the mean of fluorescenceintensity (MFI) of the PE-conjugated anti-human IgG Fcγfragment-specific F(ab′)2 goat IgG fragment. On the x-axis the testedconcentration of the different targeted single chain trimeric 4-1BBligand Fc (kih) fusion antigen binding molecules are indicated.

In FIGS. 14A and 14B it is shown that FAP-targeted 4-1BB ligandtrimer-containing Fc(kih) fusion antigen binding molecules (compound 51,filled circles) or DP47-containing 4-1BB ligand trimer-containingFc(kih) fusion antigen binding molecules (Control 1; open circles) donot bind to mouse 4-1BB expressing activated mouse splenocytes. Ananti-mouse CD137-specific human IgG1 P329G LALA antibody (clone Lob12.3)was used as positive control (triangles). The binding is characterizedby plotting the MFI of R-PE-labeled anti-human IgG Fcγ-specific goat IgGF(ab′)₂ fragment that is used as secondary detection antibody versus theconcentration in nM of the tested primary 4-1BBL trimer antigen bindingmolecules. MFI was measured by flow cytometry and baseline corrected bysubtracting the MFI of the blank control.

The scheme in FIG. 15 illustrates the general principal of the NFκBactivity assay described in Example 11.1.2. using a reporter cell line.The ratio of anti-hu IgG1 Fcγ-specific antibody to human 4-1BB ligandtrimer-containing Fc(kih) fusion antigen binding molecules was 1 to 4.

In FIG. 16 it is shown the activation of NFκB signaling pathway in thereporter cell line by secondary-antibody-crosslinked FAP-targeted singlechain trimeric 4-1BBL antigen binding molecules (Compound S1, filledcircles) or secondary-antibody-crosslinked DP47-containing single chaintrimeric 4-1BBL molecules (Control 1, open circle, dotted line). Thereporter cells were cultured for 6 hours in the presence of single chain4-1BBL trimer-containing antigen binding molecules at the indicatedconcentrations together with crosslinking secondary poly-clonalanti-huIgG1 Fcγ-specific goat IgG F(ab)₂ fragment in a 1 to 4 ratio.Luciferase activity was assessed as described in Example 11.1.1.Activity is characterized by blotting the units of released light (URL)measured during 0.5 s versus the concentration in nM of tested singlechain human 4-1BBL trimer antigen binding molecules. URLs are emitteddue to luciferase-mediated oxidation of luciferin to oxyluciferin.

The scheme in FIG. 17 illustrates the general principal of theNFκB-mediated luciferase-activity assay as described in Example 11.1.3.using HeLa-hu4-1BB-NFkB-luc clone 26 as reporter cell line. The usedratio of FAP-expressing tumor cells to human 4-1BB expressing reportercells was 5 to 1.

FIGS. 18A-D show the activation of the NFκB signaling pathway in thereporter cells via FAP-expressing tumor cell line crosslinkedFAP-targeted single chain trimeric 4-1BBL Fc(kih) fusion antigen bindingmolecules (Compound S1, filled circles). The data for not-crosslinkedDP47-containing single chain trimeric 4-1BBL molecules is shown as anegative control (Control 1, open circles, dotted line). The reportercells were cultured for 6 hours in the presence of titrated FAP-targetedor DP47-containing single chain trimeric 4-1BBL molecules andcrosslinking FAP+ tumor cell lines. The ratio between reporter cells andFAP-expressing tumor cells was 1 to 5. NFκB-mediated luciferase activityis characterized by blotting the units of released light (URL) measuredduring 0.5 s versus the concentration in nM of tested single chaintrimeric 4-1BBL antigen binding molecules. URLs are emitted due toluciferase-mediated oxidation of luciferin to oxyluciferin. Values arebaseline corrected by subtracting the URLs of the blank control. FIG.18A shows the activation with MV3 cell line as target cells, cell lineNIH/3T3-human FAP clone 39 was used as target cells in FIG. 18B. FIG.18C shows the activation with WM-266-4 cell lines as target cells andFIG. 18D shows the measured data if no FAP+ targeting cells are present.

FIGS. 19A-C show the activation of NK-KB via 4-1BB activation mediatedby FAP-targeted crosslinking of single chain trimeric 4-1BB ligand Fc(kih) fusion antigen binding molecules. Shown is the activation of NF-κBsignaling pathway in the HeLa-hu4-1BB-NFkB-luc clone 26 reporter cellsvia FAP-expressing tumor cell line crosslinked FAP-targeted single chaintrimeric 4-1BB ligand Fc (kih) fusion molecules. DP47-untargeted singlechain trimeric 4-1BB ligand Fc (kih) fusion molecules do not induceNK-κB activation (Controls 1 and 2). Monovalent FAP-targeted singlechain trimeric 4-1BB ligand Fc (kih) fusion antigen binding molecules(Compounds S1 and S3) induce a stronger NF-KB activation than bivalentFAP-targeted single chain trimeric 4-1BB ligand Fc (kih) fusion molecule(Compound S2). The HeLa-hu4-1BB-NF-κB-luc reporter cells were culturedfor 6 hours in the presence of titrated FAP-targeted or DP47-untargetedsingle chain trimeric 4-1BB ligand Fc (kih) fusion antigen bindingmolecules and crosslinking FAP+ tumor cell lines. The ratio betweenreporter cells and FAP-expressing tumor cells was 1:5. NF-κB-mediatedluciferase activity is characterized by blotting the units of releasedlight (URL) measured during 0.5 s versus the concentration in nM oftested single chain trimeric 4-1BB ligands. URLs are emitted due toluciferase-mediated oxidation of luciferin to oxyluciferin. Values arebaseline corrected by subtracting the URLs of the blank control. FIG.19A shows the activation with MV3 cell line as target cells and FIG. 19Bthe activation with WM-266-4 cell lines as target cells. FIG. 19C showsthe measured data if no FAP+ targeting cells are present.

In FIGS. 20A-B it is shown the activation of NF-KB signaling pathway inthe HeLa-hu4-1BB-NFkB-luc clone 26 reporter cells via CEA-expressingtumor cell line crosslinked CEA (sm9b)-targeted single chain trimeric4-1BB ligand Fc (kih) fusion antigen binding molecules (Compounds S4 andS5). DP47-untargeted single chain trimeric 4-1BB ligand Fc (kih) fusionmolecules do not induce NK-κB activation (Control 1 and 2). MonovalentCEA (sm9b)-targeted single chain trimeric 4-1BB ligand Fc (kih) fusionantigen molecules (compound S5) induce a stronger NF-κB activation thanbivalent CEA (sm9b)-targeted single chain trimeric 4-1BB ligand Fc (kih)fusion antigen binding molecules (compound S4). TheHeLa-hu4-1BB-NF-κB-luc reporter cells were cultured for 6 hours in thepresence of titrated CEA (Sm9b)-targeted or DP47-untargeted single chaintrimeric 4-1BB ligand Fc (kih) fusion molecules and crosslinking CEA+MKN45 tumor cell line. The ratio between reporter cells andCEA-expressing MKN45 tumor cells was 1:5. NF-κB-mediated luciferaseactivity is characterized by blotting the units of released light (URL)measured during 0.5 s versus the concentration in nM of tested singlechain trimeric 4-1BB ligands. URLs are emitted due toluciferase-mediated oxidation of luciferin to oxyluciferin. Values arebaseline corrected by subtracting the URLs of the blank control. FIG.20A shows the activation with MKN45 cell line as target cells and FIG.20B shows the measured data if no CEA+ targeting cells are present.

The activation of the NFKB signaling pathway in theHeLa-huOX40-NFkB-luc1 reporter cells via FAP-expressing WM266-4 tumorcell line crosslinking FAP-targeted single chain trimeric OX40Lmolecules (filled circles, Compounds S10) is shown in FIG. 21B. Notcrosslinked DP47-containing single chain trimeric OX40L molecules (opencircle, dotted line, Control 3) are shown as a negative control. Thereporter cells were cultured for 6 hours in the presence of titratedFAP-targeted or DP47-containing single chain trimeric OX40L moleculesand crosslinking FAP+ tumor cell lines. The ratio between reporter cellsand FAP-expressing tumor cells was 1 to 2.5. NFκB-mediated luciferaseactivity is characterized by blotting the units of released light (URL)measured during 0.5 s versus the concentration in nM of tested singlechain trimeric OX40L antigen binding molecules. URLs are emitted due toluciferase-mediated oxidation of luciferin to oxyluciferin. Values arebaseline corrected by subtracting the URLs of the blank control. Asshown in FIG. 21A, FAP-targeted single chain trimeric OX40L molecules(Compound S10, filled circles) and DP47-containing single chain trimericOX40L antigen binding molecules (Control 3) already induce a certainamount of NFκB activation if no FAP+ targeting cells are present andthus no cross-linking happened.

The scheme in FIG. 22 illustrates the general principal of theantigen-specific CD8+ T-cell activation assay described in Example10.2.2.

FIGS. 23A-F and 24A-F show the effect of single chain trimeric 4-1BBLantigen binding molecules on CD137 expression (FIGS. 23A-F) ofNLV-specific CD8+ T cells on IFNγ secretion of NLV-specific CD8+ T cells(FIGS. 24A-F). Prolonged IFNγ secretion and CD137 expression ofNLV-specific CD8⁺ T cells is strictly dependent on simultaneousactivation of T-cells via recognition of NLV-HLA-A2 complexes (signal 1,Figures A and B) and 4-1BB-triggering by FAP-targeted single chaintrimeric 4-1BBL antigen binding molecules (signal 2, Figures C to F).Filled circles: FAP-targeted single chain trimeric 4-1BBL molecules(compound S1); open circles: DP47-containing single chain trimeric4-1BBL molecules (control 3). The effect of 4-1BB upregulation is shownin FIGS. 23A to 23F, whereas the effect of INFγ expression of CD8+ Tcells is presented in FIGS. 24A to 24F. Shown is always the median offluorescence intensity (left panels) and the frequency in percentage ofpositive cells in the total CD8⁺ T cell population (right panels).

In FIGS. 25A-F and 26A-F is shown the prolonged IFNy secretion and CD137expression of NLV-specific CD8⁺ T cells that is strictly dependent onsimultaneous activation of T-cells via recognition of NLV-HLA-A2complexes (signal 1, Figures A and B) and 4-1BB-triggering byFAP-targeted single chain trimeric 4-1BB ligand (signal 2, Figures C toF). The effect of 4-1BB upregulation is shown in FIGS. 25A to 25F,whereas the effect of INFγ expression is shown in FIGS. 26A to 26F.Shown is always the median of fluorescence intensity (left panels) andthe frequency in percentage of positive cells in the total CD8+ T cellpopulation (right panels) versus the used concentration of monovalent orbivalent FAP (28H1 or 4B9)-targeted or DP47-untargeted single chaintrimeric 4-1BB ligand (kih) fusion antigen binding molecules.

FIGS. 27A-F demonstrate that costimulation with DP47-containing trimericsingle chain OX40L molecules (open circle, dotted line, Control 3) onlyslightly promoted proliferation and induced an enhanced activatedphenotype (CD25 expression) in human CD4⁺ (left side) and CD8⁺ T cells(right side) stimulated suboptimally with anti-human CD3 antibody.Hyper-crosslinking of the FAP-targeted trimeric single chain Ox40Lmolecules by the present NIH/3T3-huFAP clone 39 cells strongly increasedthis effect (filled circle, Compound S10). Shown is either the eventcount, the percentage of proliferating (CFSE-low) cells or the MFI ofCD25-PerCPCy5.5 on vital CD4⁺ and CD8⁺ T cells. Baseline values of blanksamples containing only anti-human CD3 (clone V9, huIgG1), resting humanPBMC and NIH/3T3-huFAP clone 39) were subtracted from test compoundsamples. Thus the enhancing effect of OX40L co-stimulation but not theeffect of suboptimal anti-CD3 stimulation per se is shown here.

FIG. 28A shows a polypeptide comprising three ectodomains of 4-1BBL thatare connected to each other by peptide linkers. FIG. 28B shows arepresentative example of a bivalent TNF ligand trimer-containingantigen binding molecule of the invention comprising the polypeptide ofFIG. 28A. The three TNF ligands are symbolized by the grey areas at thebottom. The white areas with black points symbolize the Fc part, thethick black point stands for the knob-into-hole modification.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as generally used in the art to which thisinvention belongs. For purposes of interpreting this specification, thefollowing definitions will apply and whenever appropriate, terms used inthe singular will also include the plural and vice versa.

As used herein, the term “antigen binding molecule” refers in itsbroadest sense to a molecule that specifically binds an antigenicdeterminant. Examples of antigen binding molecules are antibodies,antibody fragments and scaffold antigen binding proteins.

As used herein, the term “moiety capable of specific binding to a targetcell antigen” refers to a polypeptide molecule that specifically bindsto an antigenic determinant. In one aspect, the antigen binding moietyis able to activate signaling through its target cell antigen. In aparticular aspect, the antigen binding moiety is able to direct theentity to which it is attached (e.g. the TNF family ligand trimer) to atarget site, for example to a specific type of tumor cell or tumorstroma bearing the antigenic determinant. Moieties capable of specificbinding to a target cell antigen include antibodies and fragmentsthereof as further defined herein. In addition, moieties capable ofspecific binding to a target cell antigen include scaffold antigenbinding proteins as further defined herein, e.g. binding domains whichare based on designed repeat proteins or designed repeat domains (seee.g. WO 2002/020565).

In relation to an antibody or fragment thereof, the term “moiety capableof specific binding to a target cell antigen” refers to the part of themolecule that comprises the area which specifically binds to and iscomplementary to part or all of an antigen. A moiety capable of specificantigen binding may be provided, for example, by one or more antibodyvariable domains (also called antibody variable regions). Particularly,a moiety capable of specific antigen binding comprises an antibody lightchain variable region (VL) and an antibody heavy chain variable region(VH).

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, monospecific and multispecificantibodies (e.g., bispecific antibodies), and antibody fragments so longas they exhibit the desired antigen-binding activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g. containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen.

The term “monospecific” antibody as used herein denotes an antibody thathas one or more binding sites each of which bind to the same epitope ofthe same antigen. The term “bispecific” means that the antigen bindingmolecule is able to specifically bind to at least two distinct antigenicdeterminants. Typically, a bispecific antigen binding molecule comprisestwo antigen binding sites, each of which is specific for a differentantigenic determinant. In certain embodiments the bispecific antigenbinding molecule is capable of simultaneously binding two antigenicdeterminants, particularly two antigenic determinants expressed on twodistinct cells.

The term “valent” as used within the current application denotes thepresence of a specified number of binding sites in an antigen bindingmolecule. As such, the terms “bivalent”, “tetravalent”, and “hexavalent”denote the presence of two binding sites, four binding sites, and sixbinding sites, respectively, in an antigen binding molecule.

The terms “full length antibody”, “intact antibody”, and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure.“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG-classantibodies are heterotetrameric glycoproteins of about 150,000 daltons,composed of two light chains and two heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3),also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable region (VL), also called avariable light domain or a light chain variable domain, followed by alight chain constant domain (CL), also called a light chain constantregion. The heavy chain of an antibody may be assigned to one of fivetypes, called a (IgA), δ (IgD), ϵ (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g. γ1 (IgG1), γ2 (IgG2),γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies, triabodies, tetrabodies, cross-Fab fragments; linearantibodies; single-chain antibody molecules (e.g. scFv); and singledomain antibodies. For a review of certain antibody fragments, seeHudson et al., Nat Med 9, 129-134 (2003). For a review of scFvfragments, see e.g. PlUckthun, in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos.5,571,894 and 5,587,458. For discussion of Fab and F(ab′)2 fragmentscomprising salvage receptor binding epitope residues and havingincreased in vivo half-life, see U.S. Pat. No. 5,869,046. Diabodies areantibody fragments with two antigen-binding sites that may be bivalentor bispecific, see, for example, EP 404,097; WO 1993/01161; Hudson etal., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad SciUSA 90, 6444-6448 (1993). Triabodies and tetrabodies are also describedin Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodiesare antibody fragments comprising all or a portion of the heavy chainvariable domain or all or a portion of the light chain variable domainof an antibody. In certain embodiments, a single-domain antibody is ahuman single-domain antibody (Domantis, Inc., Waltham, Mass.; see e.g.U.S. Pat. No. 6,248,516 B1). Antibody fragments can be made by varioustechniques, including but not limited to proteolytic digestion of anintact antibody as well as production by recombinant host cells (e.g. E.coli or phage), as described herein.

Papain digestion of intact antibodies produces two identicalantigen-binding fragments, called “Fab” fragments containing each theheavy- and light-chain variable domains and also the constant domain ofthe light chain and the first constant domain (CH1) of the heavy chain.As used herein, Thus, the term “Fab fragment” refers to an antibodyfragment comprising a light chain fragment comprising a VL domain and aconstant domain of a light chain (CL), and a VH domain and a firstconstant domain (CH1) of a heavy chain. Fab' fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteins from theantibody hinge region. Fab′-SH are Fab′ fragments wherein the cysteineresidue(s) of the constant domains bear a free thiol group. Pepsintreatment yields an F(ab′)₂ fragment that has two antigen-combiningsites (two Fab fragments) and a part of the Fc region.

The term “cross-Fab fragment” or “xFab fragment” or “crossover Fabfragment” refers to a Fab fragment, wherein either the variable regionsor the constant regions of the heavy and light chain are exchanged. Twodifferent chain compositions of a crossover Fab molecule are possibleand comprised in the bispecific antibodies of the invention: On the onehand, the variable regions of the Fab heavy and light chain areexchanged, i.e. the crossover Fab molecule comprises a peptide chaincomposed of the light chain variable region (VL) and the heavy chainconstant region (CH1), and a peptide chain composed of the heavy chainvariable region (VH) and the light chain constant region (CL). Thiscrossover Fab molecule is also referred to as CrossFab (VLVH). On theother hand, when the constant regions of the Fab heavy and light chainare exchanged, the crossover Fab molecule comprises a peptide chaincomposed of the heavy chain variable region (VH) and the light chainconstant region (CL), and a peptide chain composed of the light chainvariable region (VL) and the heavy chain constant region (CH1). Thiscrossover Fab molecule is also referred to as CrossFab_((CLCH1)).

A “single chain Fab fragment” or “scFab” is a polypeptide consisting ofan antibody heavy chain variable domain (VH), an antibody constantdomain 1 (CH1), an antibody light chain variable domain (VL), anantibody light chain constant domain (CL) and a linker, wherein saidantibody domains and said linker have one of the following orders inN-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b)VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL;and wherein said linker is a polypeptide of at least 30 amino acids,preferably between 32 and 50 amino acids. Said single chain Fabfragments are stabilized via the natural disulfide bond between the CLdomain and the CH1 domain. In addition, these single chain Fab moleculesmight be further stabilized by generation of interchain disulfide bondsvia insertion of cysteine residues (e.g. position 44 in the variableheavy chain and position 100 in the variable light chain according toKabat numbering).

A “crossover single chain Fab fragment” or “x-scFab” is a is apolypeptide consisting of an antibody heavy chain variable domain (VH),an antibody constant domain 1 (CH1), an antibody light chain variabledomain (VL), an antibody light chain constant domain (CL) and a linker,wherein said antibody domains and said linker have one of the followingorders in N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH1 andb) VL-CH1-linker-VH-CL; wherein VH and VL form together anantigen-binding site which binds specifically to an antigen and whereinsaid linker is a polypeptide of at least 30 amino acids. In addition,these x-scFab molecules might be further stabilized by generation ofinterchain disulfide bonds via insertion of cysteine residues (e.g.position 44 in the variable heavy chain and position 100 in the variablelight chain according to Kabat numbering).

A “single-chain variable fragment (scFv)” is a fusion protein of thevariable regions of the heavy (V_(H)) and light chains (V_(L)) of anantibody, connected with a short linker peptide of ten to about 25 aminoacids. The linker is usually rich in glycine for flexibility, as well asserine or threonine for solubility, and can either connect theN-terminus of the V_(H) with the C-terminus of the V_(L), or vice versa.This protein retains the specificity of the original antibody, despiteremoval of the constant regions and the introduction of the linker. scFvantibodies are, e.g. described in

Houston, J.S., Methods in Enzymol. 203 (1991) 46-96). In addition,antibody fragments comprise single chain polypeptides having thecharacteristics of a VH domain, namely being able to assemble togetherwith a VL domain, or of a VL domain, namely being able to assembletogether with a VH domain to a functional antigen binding site andthereby providing the antigen binding property of full lengthantibodies.

“Scaffold antigen binding proteins” are known in the art, for example,fibronectin and designed ankyrin repeat proteins (DARPins) have beenused as alternative scaffolds for antigen-binding domains, see, e.g.,Gebauer and Skerra, Engineered protein scaffolds as next-generationantibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumppet al., Darpins: A new generation of protein therapeutics. DrugDiscovery Today 13: 695-701 (2008). In one aspect of the invention, ascaffold antigen binding protein is selected from the group consistingof CTLA-4 (Evibody), Lipocalins (Anticalin), a Protein A-derivedmolecule such as Z-domain of Protein A (Affibody), an A-domain(Avimer/Maxibody), a serum transferrin (trans-body); a designed ankyrinrepeat protein (DARPin), a variable domain of antibody light chain orheavy chain (single-domain antibody, sdAb), a variable domain ofantibody heavy chain (nanobody, aVH), V_(NAR) fragments, a fibronectin(AdNectin), a C-type lectin domain (Tetranectin); a variable domain of anew antigen receptor beta-lactamase (V_(NAR) fragments), a humangamma-crystallin or ubiquitin (Affilin molecules); a kunitz type domainof human protease inhibitors, microbodies such as the proteins from theknottin family, peptide aptamers and fibronectin (adnectin).

CTLA-4 (Cytotoxic T Lymphocyte-associated Antigen 4) is a CD28-familyreceptor expressed on mainly CD4+ T-cells. Its extracellular domain hasa variable domain-like Ig fold. Loops corresponding to CDRs ofantibodies can be substituted with heterologous sequence to conferdifferent binding properties. CTLA-4 molecules engineered to havedifferent binding specificities are also known as Evibodies (e.g. U.S.Pat. No. 7,166,697B1). Evibodies are around the same size as theisolated variable region of an antibody (e.g. a domain antibody). Forfurther details see Journal of Immunological Methods 248 (1-2), 31-45(2001).

Lipocalins are a family of extracellular proteins which transport smallhydrophobic molecules such as steroids, bilins, retinoids and lipids.They have a rigid beta-sheet secondary structure with a number of loopsat the open end of the conical structure which can be engineered to bindto different target antigens. Anticalins are between 160-180 amino acidsin size, and are derived from lipocalins. For further details seeBiochim Biophys Acta 1482: 337-350 (2000), U.S. Pat. No. 7,250,297B1 andUS20070224633.

An affibody is a scaffold derived from Protein A of Staphylococcusaureus which can be engineered to bind to antigen. The domain consistsof a three-helical bundle of approximately 58 amino acids. Librarieshave been generated by randomization of surface residues. For furtherdetails see Protein Eng. Des. Sel. 2004, 17, 455-462 and EP 1641818A1.

Avimers are multidomain proteins derived from the A-domain scaffoldfamily. The native domains of approximately 35 amino acids adopt adefined disulfide bonded structure. Diversity is generated by shufflingof the natural variation exhibited by the family of A-domains. Forfurther details see Nature Biotechnology 23(12), 1556-1561 (2005) andExpert Opinion on Investigational Drugs 16(6), 909-917 (June 2007).

A transferrin is a monomeric serum transport glycoprotein. Transferrinscan be engineered to bind different target antigens by insertion ofpeptide sequences in a permissive surface loop. Examples of engineeredtransferrin scaffolds include the Trans-body. For further details see J.Biol. Chem 274, 24066-24073 (1999).

Designed Ankyrin Repeat Proteins (DARPins) are derived from Ankyrinwhich is a family of proteins that mediate attachment of integralmembrane proteins to the cytoskeleton. A single ankyrin repeat is a 33residue motif consisting of two alpha-helices and a beta-turn. They canbe engineered to bind different target antigens by randomizing residuesin the first alpha-helix and a beta-turn of each repeat. Their bindinginterface can be increased by increasing the number of modules (a methodof affinity maturation). For further details see J. Mol. Biol. 332,489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369,1015-1028 (2007) and US20040132028A1.

A single-domain antibody is an antibody fragment consisting of a singlemonomeric variable antibody domain. The first single domains werederived from the variable domain of the antibody heavy chain fromcamelids (nanobodies or V_(H)H fragments). Furthermore, the termsingle-domain antibody includes an autonomous human heavy chain variabledomain (aVH) or V_(NAR) fragments derived from sharks.

Fibronectin is a scaffold which can be engineered to bind to antigen.Adnectins consists of a backbone of the natural amino acid sequence ofthe 10th domain of the 15 repeating units of human fibronectin type III(FN3). Three loops at one end of the .beta.-sandwich can be engineeredto enable an Adnectin to specifically recognize a therapeutic target ofinterest. For further details see Protein Eng. Des. Sel. 18, 435-444(2005), US20080139791, WO2005056764 and U.S. Pat. No. 6,818,418B1.

Peptide aptamers are combinatorial recognition molecules that consist ofa constant scaffold protein, typically thioredoxin (TrxA) which containsa constrained variable peptide loop inserted at the active site. Forfurther details see Expert Opin. Biol. Ther. 5, 783-797 (2005).

Microbodies are derived from naturally occurring microproteins of 25-50amino acids in length which contain 3-4 cysteine bridges—examples ofmicroproteins include KalataBI and conotoxin and knottins. Themicroproteins have a loop which can be engineered to include upto 25amino acids without affecting the overall fold of the microprotein. Forfurther details of engineered knottin domains, see WO2008098796.

An “antigen binding molecule that binds to the same epitope” as areference molecule refers to an antigen binding molecule that blocksbinding of the reference molecule to its antigen in a competition assayby 50% or more, and conversely, the reference molecule blocks binding ofthe antigen binding molecule to its antigen in a competition assay by50% or more.

The term “antigen binding domain” refers to the part of an antigenbinding molecule that comprises the area which specifically binds to andis complementary to part or all of an antigen. Where an antigen islarge, an antigen binding molecule may only bind to a particular part ofthe antigen, which part is termed an epitope. An antigen binding domainmay be provided by, for example, one or more variable domains (alsocalled variable regions). Preferably, an antigen binding domaincomprises an antibody light chain variable region (VL) and an antibodyheavy chain variable region (VH).

As used herein, the term “antigenic determinant” is synonymous with“antigen” and “epitope,” and refers to a site (e.g. a contiguous stretchof amino acids or a conformational configuration made up of differentregions of non-contiguous amino acids) on a polypeptide macromolecule towhich an antigen binding moiety binds, forming an antigen bindingmoiety-antigen complex. Useful antigenic determinants can be found, forexample, on the surfaces of tumor cells, on the surfaces ofvirus-infected cells, on the surfaces of other diseased cells, on thesurface of immune cells, free in blood serum, and/or in theextracellular matrix (ECM). The proteins useful as antigens herein canbe any native form the proteins from any vertebrate source, includingmammals such as primates (e.g. humans) and rodents (e.g. mice and rats),unless otherwise indicated. In a particular embodiment the antigen is ahuman protein. Where reference is made to a specific protein herein, theterm encompasses the “full-length”, unprocessed protein as well as anyform of the protein that results from processing in the cell. The termalso encompasses naturally occurring variants of the protein, e.g.splice variants or allelic variants.

By “specific binding” is meant that the binding is selective for theantigen and can be discriminated from unwanted or non-specificinteractions. The ability of an antigen binding molecule to bind to aspecific antigen can be measured either through an enzyme-linkedimmunosorbent assay (ELISA) or other techniques familiar to one of skillin the art, e.g. Surface Plasmon Resonance (SPR) technique (analyzed ona BlAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)),and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

In one embodiment, the extent of binding of an antigen binding moleculeto an unrelated protein is less than about 10% of the binding of theantigen binding molecule to the antigen as measured, e.g. by SPR. Incertain embodiments, an molecule that binds to the antigen has adissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸M to 10⁻¹³M,e.g. from 10⁻⁹M to 10⁻¹³ M).

“Affinity” or “binding affinity” refers to the strength of the sum totalof non-covalent interactions between a single binding site of a molecule(e.g. an antibody) and its binding partner (e.g. an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g. antibody and antigen). The affinity of amolecule X for its partner Y can generally be represented by thedissociation constant (Kd), which is the ratio of dissociation andassociation rate constants (koff and kon, respectively). Thus,equivalent affinities may comprise different rate constants, as long asthe ratio of the rate constants remains the same. Affinity can bemeasured by common methods known in the art, including those describedherein. A particular method for measuring affinity is Surface PlasmonResonance (SPR).

A “target cell antigen” as used herein refers to an antigenicdeterminant presented on the surface of a target cell, for example acell in a tumor such as a cancer cell or a cell of the tumor stroma. Incertain embodiments, the target cell antigen is an antigen on thesurface of a tumor cell. In one embodiment, target cell antigen isselected from the group consisting of Fibroblast Activation Protein(FAP), Carcinoembryonic Antigen (CEA), Melanoma-associated Chondroitin

Sulfate Proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR),CD19, CD20 and CD33. In particular, the target cell antigen isFibroblast Activation Protein (FAP).

The term “Fibroblast activation protein (FAP)”, also known as Prolylendopeptidase FAP or Seprase (EC 3.4.21), refers to any native FAP fromany vertebrate source, including mammals such as primates (e.g. humans)non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice andrats), unless otherwise indicated. The term encompasses “full-length,”unprocessed FAP as well as any form of FAP which results from processingin the cell. The term also encompasses naturally occurring variants ofFAP, e.g., splice variants or allelic variants. In one embodiment, theantigen binding molecule of the invention is capable of specific bindingto human, mouse and/or cynomolgus FAP. The amino acid sequence of humanFAP is shown in UniProt (www.uniprot.org) accession no. Q12884 (version149, SEQ ID NO:21), or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_004451.2.The extracellular domain (ECD) of human FAP extends from amino acidposition 26 to 760. The amino acid and nucleotide sequences of aHis-tagged human FAP ECD is shown in SEQ ID NOs 22 and 23, respectively.The amino acid sequence of mouse FAP is shown in UniProt accession no.P97321 (version 126, SEQ ID NO:24), or NCBI RefSeq NP_032012.1. Theextracellular domain (ECD) of mouse FAP extends from amino acid position26 to 761. SEQ ID NOs 25 and 26 show the amino acid and nucleotidesequences, respectively, of a His-tagged mouse FAP ECD. SEQ ID NOs 27and 28 show the amino acid and nucleotide sequences, respectively, of aHis-tagged cynomolgus FAP ECD. Preferably, an anti-FAP binding moleculeof the invention binds to the extracellular domain of FAP. Exemplaryanti-FAP binding molecules are described in International PatentApplication No. WO 2012/020006 A2.

The term “Carcinoembroynic antigen (CEA)”, also known asCarcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5),refers to any native CEA from any vertebrate source, including mammalssuch as primates (e.g. humans) non-human primates (e.g. cynomolgusmonkeys) and rodents (e.g. mice and rats), unless otherwise indicated.The amino acid sequence of human CEA is shown in UniProt accession no.P06731 (version 151, SEQ ID NO:29). CEA has long been identified as atumor-associated antigen (Gold and Freedman, J Exp Med., 121:439-462,1965; Berinstein N. L., J Clin Oncol., 20:2197-2207, 2002). Originallyclassified as a protein expressed only in fetal tissue, CEA has now beenidentified in several normal adult tissues. These tissues are primarilyepithelial in origin, including cells of the gastrointestinal,respiratory, and urogential tracts, and cells of colon, cervix, sweatglands, and prostate (Nap et al., Tumour Biol., 9(2-3):145-53, 1988; Napet al., Cancer Res., 52(8):2329-23339, 1992). Tumors of epithelialorigin, as well as their metastases, contain CEA as a tumor associatedantigen. While the presence of CEA itself does not indicatetransformation to a cancerous cell, the distribution of CEA isindicative. In normal tissue, CEA is generally expressed on the apicalsurface of the cell (Hammarstrom S., Semin Cancer Biol. 9(2):67-81(1999)), making it inaccessible to antibody in the blood stream. Incontrast to normal tissue, CEA tends to be expressed over the entiresurface of cancerous cells (Hammarstrom S., Semin Cancer Biol.9(2):67-81 (1999)). This change of expression pattern makes CEAaccessible to antibody binding in cancerous cells. In addition, CEAexpression increases in cancerous cells. Furthermore, increased CEAexpression promotes increased intercellular adhesions, which may lead tometastasis (Marshall J., Semin Oncol., 30(a Suppl. 8):30-6, 2003). Theprevalence of CEA expression in various tumor entities is generally veryhigh. In concordance with published data, own analyses performed intissue samples confirmed its high prevalence, with approximately 95% incolorectal carcinoma (CRC), 90% in pancreatic cancer, 80% in gastriccancer, 60% in non-small cell lung cancer (NSCLC, where it isco-expressed with HER3), and 40% in breast cancer; low expression wasfound in small cell lung cancer and glioblastoma. CEA is readily cleavedfrom the cell surface and shed into the blood stream from tumors, eitherdirectly or via the lymphatics. Because of this property, the level ofserum CEA has been used as a clinical marker for diagnosis of cancersand screening for recurrence of cancers, particularly colorectal cancer(Goldenberg D M., The International Journal of Biological Markers,7:183-188, 1992; Chau I., et al., J Clin Oncol., 22:1420-1429, 2004;Flamini et al., Clin Cancer Res; 12(23):6985-6988, 2006).

The term “Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP)”,also known as Chondroitin Sulfate Proteoglycan 4 (CSPG4) refers to anynative MCSP from any vertebrate source, including mammals such asprimates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) androdents (e.g. mice and rats), unless otherwise indicated. The amino acidsequence of human MCSP is shown in UniProt accession no. Q6UVK1 (version103, SEQ ID NO:30). The term “Epidermal Growth Factor Receptor (EGFR)”,also named Proto-oncogene c-ErbB-1 or Receptor tyrosine-protein kinaseerbB-1, refers to any native EGFR from any vertebrate source, includingmammals such as primates (e.g. humans) non-human primates (e.g.cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwiseindicated. The amino acid sequence of human EGFR is shown in UniProtaccession no. P00533 (version 211, SEQ ID NO:31).

The term “CD19” refers to B-lymphocyte antigen CD19, also known asB-lymphocyte surface antigen B4 or T-cell surface antigen Leu-12 andincludes any native CD19 from any vertebrate source, including mammalssuch as primates (e.g. humans) non-human primates (e.g. cynomolgusmonkeys) and rodents (e.g. mice and rats), unless otherwise indicated.The amino acid sequence of human CD19 is shown in Uniprot accession no.P15391 (version 160, SEQ ID NO:32). The term encompasses “full-length”unprocessed human CD19 as well as any form of human CD19 that resultsfrom processing in the cell as long as the antibody as reported hereinbinds thereto. CD19 is a structurally distinct cell surface receptorexpressed on the surface of human B cells, including, but not limitedto, pre-B cells, B cells in early development {i.e., immature B cells),mature B cells through terminal differentiation into plasma cells, andmalignant B cells. CD19 is expressed by most pre-B acute lymphoblasticleukemias (ALL), non-Hodgkin's lymphomas, B cell chronic lymphocyticleukemias (CLL), pro-lymphocytic leukemias, hairy cell leukemias, commonacute lymphocytic leukemias, and some Null-acute lymphoblasticleukemias. The expression of CD19 on plasma cells further suggests itmay be expressed on differentiated B cell tumors such as multiplemyeloma. Therefore, the CD19 antigen is a target for immunotherapy inthe treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemiaand/or acute lymphoblastic leukemia.

“CD20” refers to B-lymphocyte antigen CD20, also known asmembrane-spanning 4-domains subfamily A member 1 (MS4A1), B-lymphocytesurface antigen B1 or Leukocyte surface antigen Leu-16, and includes anynative CD20 from any vertebrate source, including mammals such asprimates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) androdents (e.g. mice and rats), unless otherwise indicated. The amino acidsequence of human CD20 is shown in Uniprot accession no. P11836 (version149, SEQ ID NO:33). “CD33” refers to Myeloid cell surface antigen CD33,also known as SIGLEC3 or gp67, and includes any native CD33 from anyvertebrate source, including mammals such as primates (e.g. humans)non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice andrats), unless otherwise indicated. The amino acid sequence of human CD33is shown in Uniprot accession no. P20138 (version 157, SEQ ID NO:34).

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding the antigenbinding molecule to antigen. The variable domains of the heavy chain andlight chain (VH and VL, respectively) of a native antibody generallyhave similar structures, with each domain comprising four conservedframework regions (FRs) and three hypervariable regions (HVRs). See,e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page91 (2007). A single VH or VL domain may be sufficient to conferantigen-binding specificity.

The term “hypervariable region” or “HVR,” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) Hypervariable regions(HVRs) are also referred to as complementarity determining regions(CDRs), and these terms are used herein interchangeably in reference toportions of the variable region that form the antigen binding regions.This particular region has been described by Kabat et al., U.S. Dept. ofHealth and Human Services, “Sequences of Proteins of ImmunologicalInterest” (1983) and by Chothia et al., J. Mol. Biol. 196:901-917(1987), where the definitions include overlapping or subsets of aminoacid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orvariants thereof is intended to be within the scope of the term asdefined and used herein. The appropriate amino acid residues whichencompass the CDRs as defined by each of the above cited references areset forth below in Table A as a comparison. The exact residue numberswhich encompass a particular CDR will vary depending on the sequence andsize of the CDR. Those skilled in the art can routinely determine whichresidues comprise a particular CDR given the variable region amino acidsequence of the antibody.

TABLE A CDR Definitions¹ CDR Kabat Chothia AbM² V_(H) CDR1 31-35 26-3226-35 V_(H) CDR2 50-65 52-58 50-58 V_(H) CDR3  95-102  95-102  95-102V_(L) CDR1 24-34 26-32 24-34 V_(L) CDR2 50-56 50-52 50-56 V_(L) CDR389-97 91-96 89-97 ¹Numbering of all CDR definitions in Table A isaccording to the numbering conventions set forth by Kabat et al. (seebelow). ²“AbM” with a lowercase “b” as used in Table A refers to theCDRs as defined by Oxford Molecular's “AbM” antibody modeling software.

Kabat et al. also defined a numbering system for variable regionsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable region sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody variable region areaccording to the Kabat numbering system.

With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. CDRs also comprise“specificity determining residues,” or “SDRs,” which are residues thatcontact antigen. SDRs are contained within regions of the CDRs calledabbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2,a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633(2008).) Unless otherwise indicated, HVR residues and other residues inthe variable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

As used herein, the term “affinity matured” in the context of antigenbinding molecules (e.g., antibodies) refers to an antigen bindingmolecule that is derived from a reference antigen binding molecule,e.g., by mutation, binds to the same antigen, preferably binds to thesame epitope, as the reference antibody; and has a higher affinity forthe antigen than that of the reference antigen binding molecule.Affinity maturation generally involves modification of one or more aminoacid residues in one or more CDRs of the antigen binding molecule.Typically, the affinity matured antigen binding molecule binds to thesame epitope as the initial reference antigen binding molecule.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g. IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ϵ, γ, and μ respectively.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization. Other forms of “humanized antibodies” encompassed by thepresent invention are those in which the constant region has beenadditionally modified or changed from that of the original antibody togenerate the properties according to the invention, especially in regardto C1q binding and/or Fc receptor (FcR) binding.

A “human” antibody is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

The term “Fe domain” or “Fc region” herein is used to define aC-terminal region of an antibody heavy chain that contains at least aportion of the constant region. The term includes native sequence Fcregions and variant Fc regions. An IgG Fc region comprises an IgG CH2and an IgG CH3 domain. The “CH2 domain” of a human IgG Fc region usuallyextends from an amino acid residue at about position 231 to an aminoacid residue at about position 340. In one embodiment, a carbohydratechain is attached to the CH2 domain. The CH2 domain herein may be anative sequence CH2 domain or variant CH2 domain. The “CH3 domain”comprises the stretch of residues C-terminal to a CH2 domain in an Fcregion (i.e. from an amino acid residue at about position 341 to anamino acid residue at about position 447 of an IgG). The CH3 regionherein may be a native sequence CH3 domain or a variant CH3 domain (e.g.a CH3 domain with an introduced “protuberance” (“knob”) in one chainthereof and a corresponding introduced “cavity” (“hole”) in the otherchain thereof; see U.S. Pat. No. 5,821,333, expressly incorporatedherein by reference). Such variant CH3 domains may be used to promoteheterodimerization of two non-identical antibody heavy chains as hereindescribed. In one embodiment, a human IgG heavy chain Fc region extendsfrom Cys226, or from Pro230, to the carboxyl-terminus of the heavychain. However, the C-terminal lysine (Lys447) of the Fc region may ormay not be present. Unless otherwise specified herein, numbering ofamino acid residues in the Fc region or constant region is according tothe EU numbering system, also called the EU index, as described in Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., 1991.

The “knob-into-hole” technology is described e.g. in U.S. Pat. No.5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621(1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, themethod involves introducing a protuberance (“knob”) at the interface ofa first polypeptide and a corresponding cavity (“hole”) in the interfaceof a second polypeptide, such that the protuberance can be positioned inthe cavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine). The protuberance and cavitycan be made by altering the nucleic acid encoding the polypeptides, e.g.by site-specific mutagenesis, or by peptide synthesis. In a specificembodiment a knob modification comprises the amino acid substitutionT366W in one of the two subunits of the Fc domain, and the holemodification comprises the amino acid substitutions T366S, L368A andY407V in the other one of the two subunits of the Fc domain. In afurther specific embodiment, the subunit of the Fc domain comprising theknob modification additionally comprises the amino acid substitutionS354C, and the subunit of the Fc domain comprising the hole modificationadditionally comprises the amino acid substitution Y349C. Introductionof these two cysteine residues results in the formation of a disulfidebridge between the two subunits of the Fc region, thus furtherstabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). Thenumbering is according to EU index of Kabat et al, Sequences of Proteinsof Immunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, MD, 1991.

A “region equivalent to the Fc region of an immunoglobulin” is intendedto include naturally occurring allelic variants of the Fc region of animmunoglobulin as well as variants having alterations which producesubstitutions, additions, or deletions but which do not decreasesubstantially the ability of the immunoglobulin to mediate effectorfunctions (such as antibody-dependent cellular cytotoxicity). Forexample, one or more amino acids can be deleted from the N-terminus orC-terminus of the Fc region of an immunoglobulin without substantialloss of biological function. Such variants can be selected according togeneral rules known in the art so as to have minimal effect on activity(see, e.g., Bowie, J. U. et al., Science 247:1306-10 (1990)).

The term “effector functions” refers to those biological activitiesattributable to the Fc region of an antibody, which vary with theantibody isotype. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity (CDC), Fc receptorbinding, antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), cytokine secretion,immune complex-mediated antigen uptake by antigen presenting cells, downregulation of cell surface receptors (e.g. B cell receptor), and B cellactivation.

An “activating Fc receptor” is an Fc receptor that following engagementby an Fc region of an antibody elicits signaling events that stimulatethe receptor-bearing cell to perform effector functions. Activating Fcreceptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), andFcαRI (CD89). A particular activating Fc receptor is human FcγRIIIa (seeUniProt accession no. P08637, version 141).

The term “TNF ligand family member” or “TNF family ligand” refers to aproinflammatory cytokine. Cytokines in general, and in particular themembers of the TNF ligand family, play a crucial role in the stimulationand coordination of the immune system. At present, nineteen cyctokineshave been identified as members of the TNF (tumour necrosis factor)ligand superfamily on the basis of sequence, functional, and structuralsimilarities. All these ligands are type II transmembrane proteins witha C-terminal extracellular domain (ectodomain), N-terminal intracellulardomain and a single transmembrane domain. The C-terminal extracellulardomain, known as TNF homology domain (THD), has 20-30% amino acididentity between the superfamily members and is responsible for bindingto the receptor. The TNF ectodomain is also responsible for the TNFligands to form trimeric complexes that are recognized by their specificreceptors.

Members of the TNF ligand family are selected from the group consistingof Lymphotoxin a (also known as LTA or TNFSF1), TNF (also known asTNFSF2), LTβ (also known as TNFSF3), OX40L (also known as TNFSF4), CD40L(also known as CD154 or TNFSF5), FasL (also known as CD95L, CD178 orTNFSF6), CD27L (also known as CD70 or TNFSF7), CD30L (also known asCD153 or TNFSF8), 4-1BBL (also known as TNFSF9), TRAIL (also known asAPO2L, CD253 or TNFSF10), RANKL (also known as CD254 or TNFSF11), TWEAK(also known as TNFSF12), APRIL (also known as CD256 or TNFSF13), BAFF(also known as CD257 or TNFSF13B), LIGHT (also known as CD258 orTNFSF14), TL1A (also known as VEGI or TNFSF15), GITRL (also known asTNFSF18), EDA-Al (also known as ectodysplasin A1) and EDA-A2 (also knownas ectodysplasin A2). The term refers to any native TNF family ligandfrom any vertebrate source, including mammals such as primates (e.g.humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g.mice and rats), unless otherwise indicated. In specific embodiments ofthe invention, the TNF ligand family member is selected from the groupconsisting of 4-1BBL, OX40L, GITRL, CD70, CD30L, CD40L and LIGHT. In aparticular embodiment, the TNF ligand family member is selected from4-1BBL and OX40L.

Further information, in particular sequences, of the TNF ligand familymembers may be obtained from publically accessible databases such asUniprot (www.uniprot.org). For instance, the human TNF ligands have thefollowing amino acid sequences: human Lymphotoxin α (UniProt accessionno. P01374, SEQ ID NO:35), human TNF (UniProt accession no. P01375, SEQID NO:36), human Lymphotoxin β (UniProt accession no. Q06643, SEQ IDNO:37), human OX40L (UniProt accession no. P23510, SEQ ID NO:38), humanCD40L (UniProt accession no. P29965, SEQ ID NO:39), human FasL (UniProtaccession no. P48023, SEQ ID NO:40), human CD27L (UniProt accession no.P32970, SEQ ID NO:41), human CD30L (UniProt accession no. P32971, SEQ IDNO:42), 4-1BBL (UniProt accession no. P41273, SEQ ID NO:43), TRAIL(UniProt accession no. P50591, SEQ ID NO:44), RANKL (UniProt accessionno. 014788, SEQ ID NO:45), TWEAK (UniProt accession no. 043508, SEQ IDNO:46), APRIL (UniProt accession no. O75888, SEQ ID NO:47), BAFF(UniProt accession no. Q9Y275, SEQ ID NO:48), LIGHT (UniProt accessionno. O43557, SEQ ID NO:49), TL1A (UniProt accession no. O95150, SEQ IDNO:50), GITRL (UniProt accession no. Q9UNG2, SEQ ID NO:51) andectodysplasin A (UniProt accession no. Q92838, SEQ ID NO:52).

An “ectodomain” is the domain of a membrane protein that extends intothe extracellular space (i.e. the space outside the target cell).Ectodomains are usually the parts of proteins that initiate contact withsurfaces, which leads to signal transduction. The ectodomain of TNFligand family member as defined herein thus refers to the part of theTNF ligand protein that extends into the extracellular space (theextracellular domain), but also includes shorter parts or fragmentsthereof that are responsible for the trimerization and for the bindingto the corresponding TNF receptor. The term “ectodomain of a TNF ligandfamily member or a fragment thereof” thus refers to the extracellulardomain of the TNF ligand family member that forms the extracellulardomain or to parts thereof that are still able to bind to the receptor(receptor binding domain).

The term “costimulatory TNF ligand family member” or “costimulatory TNFfamily ligand” refers to a subgroup of TNF ligand family members, whichare able to costimulate proliferation and cytokine production ofT-cells. These TNF family ligands can costimulate TCR signals uponinteraction with their corresponding TNF receptors and the interactionwith their receptors leads to recruitment of TNFR-associated factors(TRAF), which initiate signalling cascades that result in T-cellactivation. Costimulatory TNF family ligands are selected from the groupconsisting of 4-1BBL, OX40L, GITRL, CD70, CD30L and LIGHT, moreparticularly the costimulatory TNF ligand family member is selected from4-1BBL and OX40L.

As described herein before, 4-1BBL is a type II transmembrane proteinand one member of the TNF ligand family. Complete or full length 4-1BBLhaving the amino acid sequence of SEQ ID NO:43 has been described toform trimers on the surface of cells. The formation of trimers isenabled by specific motives of the ectodomain of 4-1BBL. Said motivesare designated herein as “trimerization region”. The amino acids 50-254of the human 4-1BBL sequence (SEQ ID NO:53) form the extracellulardomain of 4-1BBL, but even fragments thereof are able to form thetrimers.

In specific embodiments of the invention, the term “ectodomain of 4-1BBLor a fragment thereof” refers to a polypeptide having an amino acidsequence selected from SEQ ID NO:4 (amino acids 52-254 of human 4-1BBL),SEQ ID NO:1 (amino acids 71-254 of human 4-1BBL), SEQ ID NO:3 (aminoacids 80-254 of human 4-1BBL), SEQ ID NO:2 (amino acids 85-254 of human4-1BBL), SEQ ID NO:99 (amino acids 71-248 of human 4-1BBL), SEQ IDNO:100 (amino acids 85-248 of human 4-1BBL), SEQ ID NO:101 (amino acids80-248 of human 4-1BBL) and SEQ ID NO:102 (amino acids 52-254 of human4-1BBL), but also other fragments of the ectodomain capable oftrimerization are included herein.

As described herein before, OX40L is another type II transmembraneprotein and a further member of the TNF ligand family. Complete or fulllength human OX40L has the amino acid sequence of SEQ ID NO:38. Theamino acids 51-183 of the human OX40L sequence (SEQ ID NO:6) form theextracellular domain of OX40L, but even fragments thereof that are ableto form the trimers. In specific embodiments of the invention, the term“ectodomain of OX40L or a fragment thereof” refers to a polypeptidehaving an amino acid sequence selected from SEQ ID NO:6 (amino acids51-183 of human OX40L) or SEQ ID NO:7 (amino acids 52-183 of humanOX40L), but also other fragments of the ectodomain capable oftrimerization are included herein.

The term “peptide linker” refers to a peptide comprising one or moreamino acids. A peptide linker comprises 1 to 44 amino acids, moreparticularly 2 to 20 amino acids. Peptide linkers are known in the artor are described herein. Suitable, non-immunogenic linker peptides are,for example, (G₄S)_(n), (SG₄)_(n) or G₄(SG₄)_(n) peptide linkers,wherein “n” is generally a number between 1 and 10, typically between 2and 4, in particular 2, i.e. the peptides selected from the groupconsisting of GGGGS (SEQ ID NO:136), GGGGSGGGGS (SEQ ID NO:54),SGGGGSGGGG (SEQ ID NO:55) and GGGGSGGGGSGGGG (SEQ ID NO:56), but alsoinclude the sequences GSPGSSSSGS (SEQ ID NO:57), GSGSGSGS (SEQ IDNO:58), GSGSGNGS (SEQ ID NO:59), GGSGSGSG (SEQ ID NO:60), GGSGSG (SEQ IDNO:61), GGSG (SEQ ID NO:62), GGSGNGSG (SEQ ID NO:63), GGNGSGSG (SEQ IDNO:64) and GGNGSG (SEQ ID NO:65). Peptide linkers of particular interestare ((G4S)₁ or GGGGS (SEQ ID NO:136), (G₄5)₂ or GGGGSGGGGS (SEQ IDNO:54) and GSPGSSSSGS (SEQ ID NO:57), more particularly (G₄5)₂ orGGGGSGGGGS (SEQ ID NO:54) and GSPGSSSSGS (SEQ ID NO:57).

The term “amino acid” as used within this application denotes the groupof naturally occurring carboxy α-amino acids comprising alanine (threeletter code: ala, one letter code: A), arginine (arg, R), asparagine(asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q),glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine(ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M),phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine(thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

A “single chain fusion protein” as used herein refers to a single chainpolypeptide composed of one or two ectodomains of said TNF ligand familymember fused to a part of antigen binding moiety or Fc part. The fusionmay occur by directly linking the N or C-terminal amino acid of theantigen binding moiety via a peptide linker to the C- or N-terminalamino acid of the ectodomain of said TNF ligand family member.

By “fused” or “connected” is meant that the components (e.g. apolypeptide and an ectodomain of said TNF ligand family member) arelinked by peptide bonds, either directly or via one or more peptidelinkers.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide (protein) sequence is defined as the percentage of aminoacid residues in a candidate sequence that are identical with the aminoacid residues in the reference polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN. SAWIor Megalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for aligning sequences, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the sequence comparisoncomputer program ALIGN-2. The ALIGN-2 sequence comparison computerprogram was authored by Genentech, Inc., and the source code has beenfiled with user documentation in the U.S. Copyright Office, WashingtonD.C., 20559, where it is registered under U.S. Copyright RegistrationNo. TXU510087. The ALIGN-2 program is publicly available from Genentech,Inc., South San Francisco, Calif., or may be compiled from the sourcecode. The ALIGN-2 program should be compiled for use on a UNIX operatingsystem, including digital UNIX V4.0D. All sequence comparison parametersare set by the ALIGN-2 program and do not vary. In situations whereALIGN-2 is employed for amino acid sequence comparisons, the % aminoacid sequence identity of a given amino acid sequence A to, with, oragainst a given amino acid sequence B (which can alternatively bephrased as a given amino acid sequence A that has or comprises a certain% amino acid sequence identity to, with, or against a given amino acidsequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

In certain embodiments, amino acid sequence variants of the TNF ligandtrimer-containing antigen binding molecules provided herein arecontemplated. For example, it may be desirable to improve the bindingaffinity and/or other biological properties of the TNF ligandtrimer-containing antigen binding molecules. Amino acid sequencevariants of the TNF ligand trimer-containing antigen binding moleculesmay be prepared by introducing appropriate modifications into thenucleotide sequence encoding the molecules, or by peptide synthesis.Such modifications include, for example, deletions from, and/orinsertions into and/or substitutions of residues within the amino acidsequences of the antibody. Any combination of deletion, insertion, andsubstitution can be made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics, e.g.,antigen-binding. Sites of interest for substitutional mutagenesisinclude the HVRs and Framework (FRs). Conservative substitutions areprovided in Table B under the heading “Preferred Substitutions” andfurther described below in reference to amino acid side chain classes(1) to (6). Amino acid substitutions may be introduced into the moleculeof interest and the products screened for a desired activity, e.g.,retained/improved antigen binding, decreased immunogenicity, or improvedADCC or CDC.

TABLE B Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

The term “amino acid sequence variants” includes substantial variantswherein there are amino acid substitutions in one or more hypervariableregion residues of a parent antigen binding molecule (e.g. a humanizedor human antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antigen binding molecule and/or will havesubstantially retained certain biological properties of the parentantigen binding molecule. An exemplary substitutional variant is anaffinity matured antibody, which may be conveniently generated, e.g.,using phage display-based affinity maturation techniques such as thosedescribed herein. Briefly, one or more HVR residues are mutated and thevariant antigen binding molecules displayed on phage and screened for aparticular biological activity (e.g. binding affinity). In certainembodiments, substitutions, insertions, or deletions may occur withinone or more HVRs so long as such alterations do not substantially reducethe ability of the antigen binding molecule to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. A useful method for identification of residues orregions of an antibody that may be targeted for mutagenesis is called“alanine scanning mutagenesis” as described by Cunningham and Wells(1989) Science, 244:1081-1085. In this method, a residue or group oftarget residues (e.g., charged residues such as Arg, Asp, His, Lys, andGlu) are identified and replaced by a neutral or negatively chargedamino acid (e.g., alanine or polyalanine) to determine whether theinteraction of the antibody with antigen is affected. Furthersubstitutions may be introduced at the amino acid locationsdemonstrating functional sensitivity to the initial substitutions.Alternatively, or additionally, a crystal structure of anantigen-antigen binding molecule complex to identify contact pointsbetween the antibody and antigen. Such contact residues and neighboringresidues may be targeted or eliminated as candidates for substitution.Variants may be screened to determine whether they contain the desiredproperties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includeTNF family ligand trimer-containing antigen binding molecule with anN-terminal methionyl residue. Other insertional variants of the moleculeinclude the fusion to the N- or C-terminus to a polypeptide whichincreases the serum half-life of the TNF ligand trimer-containingantigen binding molecules.

In certain embodiments, the TNF family ligand trimer-containing antigenbinding molecules provided herein are altered to increase or decreasethe extent to which the antibody is glycosylated. Glycosylation variantsof the molecules may be conveniently obtained by altering the amino acidsequence such that one or more glycosylation sites is created orremoved. Where the TNF ligand trimer-containing antigen binding moleculecomprises an Fc region, the carbohydrate attached thereto may bealtered. Native antibodies produced by mammalian cells typicallycomprise a branched, biantennary oligosaccharide that is generallyattached by an N-linkage to Asn297 of the CH2 domain of the Fc region.See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharidemay include various carbohydrates, e.g., mannose, N-acetyl glucosamine(GlcNAc), galactose, and sialic acid, as well as a fucose attached to aGlcNAc in the “stem” of the biantennary oligosaccharide structure. Insome embodiments, modifications of the oligosaccharide in TNF familyligand trimer-containing antigen binding molecule may be made in orderto create variants with certain improved properties. In one aspect,variants of TNF family ligand trimer-containing antigen bindingmolecules are provided having a carbohydrate structure that lacks fucoseattached (directly or indirectly) to an Fc region. Such fucosylationvariants may have improved ADCC function, see e.g. US Patent PublicationNos. US 2003/0157108 (Presta, L.) or US 2004/0093621 (Kyowa Hakko KogyoCo., Ltd). Further variants of the TNF family ligand trimer-containingantigen binding molecules of the invention include those with bisectedoligosaccharides, e.g., in which a biantennary oligosaccharide attachedto the Fc region is bisected by GlcNAc. Such variants may have reducedfucosylation and/or improved ADCC function., see for example WO2003/011878 (Jean-Mairet et al.); US Pat. No. 6,602,684 (Umana et al.);and US 2005/0123546 (Umana et al.). Variants with at least one galactoseresidue in the oligosaccharide attached to the Fc region are alsoprovided. Such antibody variants may have improved CDC function and aredescribed, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju,S.); and WO 1999/22764 (Raju, S.).

In certain embodiments, it may be desirable to create cysteineengineered variants of the TNF family ligand trimer-containing antigenbinding molecule of the invention, e.g., “thioMAbs,” in which one ormore residues of the molecule are substituted with cysteine residues. Inparticular embodiments, the substituted residues occur at accessiblesites of the molecule. By substituting those residues with cysteine,reactive thiol groups are thereby positioned at accessible sites of theantibody and may be used to conjugate the antibody to other moieties,such as drug moieties or linker-drug moieties, to create animmunoconjugate. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and 5400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antigen binding molecules may be generated as described,e.g., in U.S. Pat. No. 7,521,541.

In certain aspects, the TNF family ligand trimer-containing antigenbinding molecules provided herein may be further modified to containadditional non-proteinaceous moieties that are known in the art andreadily available. The moieties suitable for derivatization of theantibody include but are not limited to water soluble polymers.Non-limiting examples of water soluble polymers include, but are notlimited to, polyethylene glycol (PEG), copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer isattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether thebispecific antibody derivative will be used in a therapy under definedconditions, etc. In another aspect, conjugates of an antibody andnon-proteinaceous moiety that may be selectively heated by exposure toradiation are provided. In one embodiment, the non-proteinaceous moietyis a carbon nanotube (Kam, N. W. et al., Proc. Natl. Acad. Sci. USA 102(2005) 11600-11605). The radiation may be of any wavelength, andincludes, but is not limited to, wavelengths that do not harm ordinarycells, but which heat the non-proteinaceous moiety to a temperature atwhich cells proximal to the antibody-non-proteinaceous moiety arekilled.

In another aspect, immunoconjugates of the TNF family ligandtrimer-containing antigen binding molecules provided herein maybeobtained. An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

The term “polynucleotide” refers to an isolated nucleic acid molecule orconstruct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmidDNA (pDNA). A polynucleotide may comprise a conventional phosphodiesterbond or a non-conventional bond (e.g. an amide bond, such as found inpeptide nucleic acids (PNA). The term “nucleic acid molecule” refers toany one or more nucleic acid segments, e.g. DNA or RNA fragments,present in a polynucleotide.

By “isolated” nucleic acid molecule or polynucleotide is intended anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encoding apolypeptide contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. An isolated polynucleotide includes apolynucleotide molecule contained in cells that ordinarily contain thepolynucleotide molecule, but the polynucleotide molecule is presentextrachromosomally or at a chromosomal location that is different fromits natural chromosomal location. Isolated RNA molecules include in vivoor in vitro RNA transcripts of the present invention, as well aspositive and negative strand forms, and double-stranded forms. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. In addition, apolynucleotide or a nucleic acid may be or may include a regulatoryelement such as a promoter, ribosome binding site, or a transcriptionterminator.

By a nucleic acid or polynucleotide having a nucleotide sequence atleast, for example, 95% “identical” to a reference nucleotide sequenceof the present invention, it is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence. In otherwords, to obtain a polynucleotide having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at the5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence. As a practical matter,whether any particular polynucleotide sequence is at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of thepresent invention can be determined conventionally using known computerprograms, such as the ones discussed above for polypeptides (e.g.ALIGN-2).

The term “expression cassette” refers to a polynucleotide generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in atarget cell. The recombinant expression cassette can be incorporatedinto a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, ornucleic acid fragment. Typically, the recombinant expression cassetteportion of an expression vector includes, among other sequences, anucleic acid sequence to be transcribed and a promoter. In certainembodiments, the expression cassette of the invention comprisespolynucleotide sequences that encode bispecific antigen bindingmolecules of the invention or fragments thereof.

The term “vector” or “expression vector” is synonymous with “expressionconstruct” and refers to a DNA molecule that is used to introduce anddirect the expression of a specific gene to which it is operablyassociated in a target cell. The term includes the vector as aself-replicating nucleic acid structure as well as the vectorincorporated into the genome of a host cell into which it has beenintroduced. The expression vector of the present invention comprises anexpression cassette. Expression vectors allow transcription of largeamounts of stable mRNA. Once the expression vector is inside the targetcell, the ribonucleic acid molecule or protein that is encoded by thegene is produced by the cellular transcription and/or translationmachinery. In one embodiment, the expression vector of the inventioncomprises an expression cassette that comprises polynucleotide sequencesthat encode bispecific antigen binding molecules of the invention orfragments thereof.

The terms “host cell”, “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages.

Progeny may not be completely identical in nucleic acid content to aparent cell, but may contain mutations. Mutant progeny that have thesame function or biological activity as screened or selected for in theoriginally transformed cell are included herein. A host cell is any typeof cellular system that can be used to generate the bispecific antigenbinding molecules of the present invention. Host cells include culturedcells, e.g. mammalian cultured cells, such as CHO cells, BHK cells, NSOcells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PERcells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, andplant cells, to name only a few, but also cells comprised within atransgenic animal, transgenic plant or cultured plant or animal tissue.

An “effective amount” of an agent refers to the amount that is necessaryto result in a physiological change in the cell or tissue to which it isadministered.

A “therapeutically effective amount” of an agent, e.g. a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g. humans and non-human primates such as monkeys),rabbits, and rodents (e.g. mice and rats). Particularly, the individualor subject is a human.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable excipient” refers to an ingredient in apharmaceutical composition, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable excipient includes,but is not limited to, a buffer, a stabilizer, or a preservative.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, the moleculesof the invention are used to delay development of a disease or to slowthe progression of a disease.

The term “cancer” as used herein refers to proliferative diseases, suchas lymphomas, carcinoma, lymphoma, blastoma, sarcoma, leukemia,lymphocytic leukemias, lung cancer, non-small cell lung (NSCL) cancer,bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer,skin cancer, cancer of the head or neck, cutaneous or intraocularmelanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of theanal region, stomach cancer, gastric cancer, colorectal cancer (CRC),pancreatic cancer, breast cancer, triple-negative breast cancer ,uterine cancer, carcinoma of the fallopian tubes, carcinoma of theendometrium, carcinoma of the cervix, carcinoma of the vagina, carcinomaof the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of thesmall intestine, cancer of the endocrine system, cancer of the thyroidgland, cancer of the parathyroid gland, cancer of the adrenal gland,sarcoma of soft tissue, cancer of the urethra, cancer of the penis,prostate cancer, cancer of the bladder, cancer of the kidney or ureter,renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma,hepatocellular cancer, biliary cancer, neoplasms of the central nervoussystem (CNS), spinal axis tumors, brain stem glioma, glioblastomamultiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas,meningiomas, squamous cell carcinomas, pituitary adenoma and Ewingssarcoma, melanoma, multiple myeloma, B-cell cancer (lymphoma), chroniclymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairycell leukemia, chronic myeloblastic leukemia, including refractoryversions of any of the above cancers, or a combination of one or more ofthe above cancers.

TNF Family Ligand Trimer-Containing Antigen Binding Molecules of theInvention

The invention provides novel TNF family ligand trimer-containing antigenbinding molecules with particularly advantageous properties such asproducibility, stability, binding affinity, biological activity,targeting efficiency and reduced toxicity.

Thus, the invention relates to a TNF family ligand trimer-containingantigen binding molecule comprising

-   (a) at least one moiety capable of specific binding to a target cell    antigen,-   (b) a polypeptide comprising three ectodomains of a TNF ligand    family member or fragments thereof that are connected to each other,    optionally by peptide linkers, and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association.

In one aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising

-   (a) at least one moiety capable of specific binding to a target cell    antigen,-   (b) a polypeptide comprising three ectodomains of a TNF ligand    family member or fragments thereof that are connected to each other    by peptide linkers and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association.

In another aspect, the TNF family ligand trimer containing antigenbinding molecule comprises

-   (a) at least one moiety capable of specific binding to a target cell    antigen,-   (b) a polypeptide comprising three ectodomains of a TNF ligand    family member or fragments thereof that are connected to each other    by peptide linkers and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association, wherein the polypeptide comprising the three    ectodomains of a TNF ligand family member or fragments thereof that    are connected to each other by peptide linkers is fused to the N- or    C-terminal amino acid of one of the two subunits of the Fc domain,    optionally through a peptide linker.

In a particular aspect, the TNF family ligand trimer-containing antigenbinding molecule comprises (a) at least one moiety capable of specificbinding to a target cell antigen, (b) a polypeptide comprising threeectodomains of a TNF ligand family member or fragments thereof that areconnected to each other by peptide linkers and (c) a Fc domain composedof a first and a second subunit capable of stable association, whereinthe TNF ligand family member costimulates human T-cell activation. Thus,the TNF ligand family member is a costimulatory TNF family ligand. Inparticular, the costimulatory TNF family ligand is selected from thegroup consisting of 4-1BBL, OX40L, GITRL, CD70, CD30L and LIGHT, moreparticularly the costimulatory TNF family ligand is selected from 4-1BBLand OX40L.

In one aspect, the TNF ligand family member is 4-1BBL.

In a further aspect, the ectodomain of a TNF ligand family membercomprises the amino acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:99, SEQID NO: 100, SEQ ID NO:101 and SEQ ID NO:102, particularly the amino acidsequence of SEQ ID NO:1 or SEQ ID NO:99.

In a particular aspect, the ectodomain of a TNF ligand family member orfragment thereof comprises the amino acid sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ IDNO:4, particularly the amino acid sequence of SEQ ID NO:1.

In a further aspect, the TNF family ligand trimer-containing antigenbinding molecule of the invention comprises

-   (a) at least one moiety capable of specific binding to a target cell    antigen and-   (b) a polypeptide comprising the amino acid sequence of SEQ ID NO:5    or SEQ ID NO:103, and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association. More particularly, the polypeptide comprises the    amino acid sequence of SEQ ID NO:5.

In another aspect, the TNF ligand family member is OX40L.

In another aspect, the ectodomain of a TNF ligand family membercomprises the amino acid sequence selected from the group consisting ofSEQ ID NO:6 and SEQ ID NO:7, particularly the amino acid sequence of SEQID NO:6.

In a further aspect, the TNF family ligand trimer-containing antigenbinding molecule of the invention comprises

-   (a) at least one moiety capable of specific binding to a target cell    antigen and-   (b) a polypeptide comprising the amino acid sequence of SEQ ID NO:8    and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association.

In one aspect, the TNF family ligand trimer-containing antigen bindingmolecule comprises (a) at least one moiety capable of specific bindingto a target cell antigen, (b) a polypeptide comprising three ectodomainsof a TNF ligand family member or fragments thereof that are connected toeach other and (c) a Fc domain composed of a first and a second subunitcapable of stable association, wherein the three ectodomains of a TNFligand family member or fragments thereof are linked to each otherdirectly, or with a suitable chemical linker, such as a disulfide, esteror ether bridge.

In another aspect, the TNF family ligand trimer-containing antigenbinding molecule comprises (a) at least one moiety capable of specificbinding to a target cell antigen, (b) a polypeptide comprising threeectodomains of a TNF ligand family member or fragments thereof that areconnected to each other by peptide linkers and (c) a Fc domain composedof a first and a second subunit capable of stable association, whereinthe three ectodomains of a TNF ligand family member or fragments thereofare linked to each other by peptide linkers that are the same ordifferent and are selected from the group consisting of GGGGSGGGGS (SEQID NO:54), SGGGGSGGGG (SEQ ID NO:55) and GGGGSGGGGSGGGG (SEQ ID NO:56),but also include the sequences GSPGSSSSGS (SEQ ID NO:57), GSGSGSGS (SEQID NO:58), GSGSGNGS (SEQ ID NO:59), GGSGSGSG (SEQ ID NO:60), GGSGSG (SEQID NO:61), GGSG (SEQ ID NO:62), GGSGNGSG (SEQ ID NO:63), GGNGSGSG (SEQID NO:64) and GGNGSG (SEQ ID NO:65). In particular, the peptide linkersare the same and are selected from the group consisting of (G₄5)₂ (SEQID NO:54) and GSPGSSSSGS (SEQ ID NO:57). More particularly, theinvention relates to a TNF family ligand trimer-containing antigenbinding molecule as defined above, wherein the three ectodomains of aTNF ligand family member or fragments thereof are connected to eachother by (G45)₂ linkers.

In another aspect, the TNF family ligand trimer-containing antigenbinding molecule of the invention comprises (a) at least one moietycapable of specific binding to a target cell antigen, (b) a polypeptidecomprising three ectodomains of a TNF ligand family member or fragmentsthereof that are connected to each other by peptide linkers and (c) a Fcdomain composed of a first and a second subunit capable of stableassociation, wherein the polypeptide comprising three ectodomains of aTNF ligand family member or fragments thereof is fused at the C-terminalamino acid to the N-terminal amino acid of one of the subunits of the Fcdomain. In a particular aspect, the polypeptide comprising threeectodomains of a TNF ligand family member or fragments thereof is fusedat the C-terminal amino acid to the N-terminal amino acid of a CH2domain in the Fc domain. In a further aspect, the polypeptide comprisingthree ectodomains of a TNF ligand family member or fragments thereof isconnected at the C-terminal amino acid by a peptide linker to theN-terminal amino acid of the Fc domain.

In another aspect, the TNF family ligand trimer-containing antigenbinding molecule of the invention comprises (a) at least one moietycapable of specific binding to a target cell antigen, (b) a polypeptidecomprising three ectodomains of a TNF ligand family member or fragmentsthereof that are connected to each other by peptide linkers and (c) a Fcdomain composed of a first and a second subunit capable of stableassociation, wherein the polypeptide comprising three ectodomains of aTNF ligand family member or fragments thereof is fused at the N-terminalamino acid to the C-terminal amino acid of the Fc domain. In aparticular aspect, the polypeptide comprising three ectodomains of a TNFligand family member or fragments thereof is fused at the N-terminalamino acid to the C-terminal amino acid of a CH3 domain in the Fcdomain. In a further aspect, the polypeptide comprising threeectodomains of a TNF ligand family member or fragments thereof isconnected at the N-terminal amino acid by a peptide linker to theC-terminal amino acid of the Fc domain.

In another aspect, the TNF family ligand trimer-containing antigenbinding molecule comprises (a) at least one moiety capable of specificbinding to a target cell antigen, (b) a polypeptide comprising threeectodomains of a TNF ligand family member or fragments thereof that areconnected to each other by peptide linkers and (c) a Fc domain composedof a first and a second subunit capable of stable association, whereinthe moiety capable of specific binding to a target cell antigen is notfused to the polypeptide comprising three ectodomains of a TNF ligandfamily member or fragments thereof.

In a further aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule as defined herein before,wherein the moiety capable of specific binding to a target cell antigenis selected from the group consisting of an antibody, an antibodyfragment and a scaffold antigen binding protein.

In one aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule as defined herein before,wherein the moiety capable of specific binding to a target cell antigenis an antibody.

In another aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule as defined herein before,wherein the moiety capable of specific binding to a target cell antigenis an antibody fragment. In particular, the antibody fragment isselected from the group consisting of a Fab molecule, a crossover Fabmolecule, a single chain Fab molecule, a Fv molecule, a scFv molecule, asingle domain antibody, and aVH.

In a further aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule as defined herein before,wherein the moiety capable of specific binding to a target cell antigenis a scaffold antigen binding protein.

Thus, in a further aspect, the invention relates to a TNF family ligandtrimer-containing antigen binding molecule as defined herein before,wherein the moiety capable of specific binding to a target cell antigenis selected from the group consisting of an antibody fragment, a Fabmolecule, a crossover Fab molecule, a single chain Fab molecule, a Fvmolecule, a scFv molecule, a single domain antibody, an aVH and ascaffold antigen binding protein. Thus, in a particular aspect, theinvention provides a TNF family ligand trimer-containing antigen bindingmolecule comprising (a) at least one antibody fragment capable ofspecific binding to a target cell antigen which is selected from thegroup consisting of a Fab molecule, a crossover Fab molecule, a singlechain Fab molecule, a Fv molecule, a scFv molecule, a single domainantibody, an aVH and a scaffold antigen binding protein, (b) apolypeptide comprising three ectodomains of a TNF ligand family memberor fragments thereof that are connected to each other by peptide linkersand (c) a Fc domain composed of a first and a second subunit capable ofstable association. In one aspect, the TNF family ligandtrimer-containing antigen binding molecule comprising (a) one antibodyfragment capable of specific binding to a target cell antigen which isselected from the group consisting of a Fab molecule, a crossover Fabmolecule, a single chain Fab molecule, a Fv molecule, a scFv molecule, asingle domain antibody, an aVH and a scaffold antigen binding protein.

In a particular aspect, the invention is concerned with a TNF familyligand trimer-containing antigen binding molecule as defined above,wherein the moiety capable of specific binding to a target cell antigenis a Fab molecule capable of specific binding to a target cell antigen.Thus, the invention provides a TNF family ligand trimer-containingantigen binding molecule comprising (a) at least one Fab moleculecapable of specific binding to a target cell antigen, (b) a polypeptidecomprising three ectodomains of a TNF ligand family member or fragmentsthereof that are connected to each other by peptide linkers and (c) a Fcdomain composed of a first and a second subunit capable of stableassociation.

In another aspect, provided is a TNF family ligand trimer-containingantigen binding molecule of the invention, wherein the target cellantigen is selected from the group consisting of Fibroblast ActivationProtein (FAP), Carcinoembryonic Antigen (CEA), Melanoma-associatedChondroitin Sulfate Proteoglycan (MCSP), Epidermal Growth FactorReceptor (EGFR), CD19, CD20 and CD33.

In a further aspect, provided is a TNF family ligand trimer-containingantigen binding molecule as described herein before, wherein the Fcdomain composed of a first and a second subunit capable of stableassociation is an IgG Fc domain, particularly an IgG1 Fc domain or anIgG4 Fc domain. In particular, the Fc domain composed of a first and asecond subunit capable of stable association is an IgG1 Fc domain. In aparticular aspect, the Fc domain comprises a modification promoting theassociation of the first and second subunit of the Fc domain.

Fc Domain Modifications Reducing Fc Receptor Binding and/or EffectorFunction

The Fc domain of the TNF family ligand trimer-containing antigen bindingmolecules of the invention consists of a pair of polypeptide chainscomprising heavy chain domains of an immunoglobulin molecule. Forexample, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer,each subunit of which comprises the CH2 and CH3 IgG heavy chain constantdomains. The two subunits of the Fc domain are capable of stableassociation with each other.

The Fc domain confers favorable pharmacokinetic properties to theantigen binding molecules of the invention, including a long serumhalf-life which contributes to good accumulation in the target tissueand a favorable tissue-blood distribution ratio. The Fc domain thusfavorably contributes to the pharmacokinetic parameters (PK data) of theantigen binding molecules, such as as clearance, volume of distributionor elimination half-time (t_(1/2)). At the same time it may, however,lead to undesirable targeting of the bispecific antibodies of theinvention to cells expressing Fc receptors rather than to the preferredantigen-bearing cells. Accordingly, in particular aspects, the Fc domainof the TNF family ligand trimer-containing antigen binding molecule ofthe invention exhibits reduced binding affinity to an Fc receptor and/orreduced effector function, as compared to a native IgG1 Fc domain. Inone aspect, the Fc does not substantially bind to an Fc receptor and/ordoes not induce effector function. In a particular aspect the Fcreceptor is an Fcy receptor. In one aspect, the Fc receptor is a humanFc receptor. In a specific aspect, the Fc receptor is an activatinghuman Fcy receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa,most specifically human FcγRIIIa. In one aspect, the Fc domain does notinduce effector function. The reduced effector function can include, butis not limited to, one or more of the following: reduced complementdependent cytotoxicity (CDC), reduced antibody-dependent cell-mediatedcytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis(ADCP), reduced cytokine secretion, reduced immune complex-mediatedantigen uptake by antigen-presenting cells, reduced binding to NK cells,reduced binding to macrophages, reduced binding to monocytes, reducedbinding to polymorphonuclear cells, reduced direct signaling inducingapoptosis, reduced dendritic cell maturation, or reduced T cell priming.

In certain aspects, one or more amino acid modifications may beintroduced into the Fc region of a TNF family ligand trimer-containingantigen binding molecule provided herein, thereby generating an Fcregion variant. The Fc region variant may comprise a human Fc regionsequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprisingan amino acid modification (e.g. a substitution) at one or more aminoacid positions.

In one aspect, the invention is concerned with a TNF family ligandtrimer-containing antigen binding molecule as defined herein before,comprising

-   (a) at least one moiety capable of specific binding to a target cell    antigen,-   (b) a polypeptide comprising three ectodomains of a TNF ligand    family member or fragments thereof that are connected to each other    by peptide linkers and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association, wherein the Fc domain comprises one or more    amino acid substitution that reduces binding to an Fc receptor, in    particular towards Fcy receptor.

In one aspect, the Fc domain of the TNF family ligand trimer-containingantigen binding molecule of the invention comprises one or more aminoacid mutation that reduces the binding affinity of the Fc domain to anFc receptor and/or effector function. Typically, the same one or moreamino acid mutation is present in each of the two subunits of the Fcdomain. In particular, the Fc domain comprises an amino acidsubstitution at a position of E233, L234, L235, N297, P331 and P329 (EUnumbering). In particular, the Fc domain comprises amino acidsubstitutions at positions 234 and 235 (EU numbering) and/or 329 (EUnumbering) of the IgG heavy chains. More particularly, provided is atrimeric TNF family ligand-containing antigen binding molecule accordingto the invention which comprises an Fc domain with the amino acidsubstitutions L234A, L235A and P329G (“P329G LALA”, EU numbering) in theIgG heavy chains. The amino acid substitutions L234A and L235A refer tothe so-called LALA mutation. The “P329G LALA” combination of amino acidsubstitutions almost completely abolishes Fcy receptor binding of ahuman IgG1 Fc domain and is described in International Patent Appl.Publ. No. WO 2012/130831 A1 which also describes methods of preparingsuch mutant Fc domains and methods for determining its properties suchas Fc receptor binding or effector functions. “EU numbering” refers tothe numbering according to EU index of Kabat et al , Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Mf., 1991.

Fc domains with reduced Fc receptor binding and/or effector functionalso include those with substitution of one or more of Fc domainresidues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056).Such Fc mutants include Fc mutants with substitutions at two or more ofamino acid positions 265, 269, 270, 297 and 327, including the so-called“DANA” Fc mutant with substitution of residues 265 and 297 to alanine(U.S. Pat. No. 7,332,581).

In another aspect, the Fc domain is an IgG4 Fc domain. IgG4 antibodiesexhibit reduced binding affinity to Fc receptors and reduced effectorfunctions as compared to IgG1 antibodies. In a more specific aspect, theFc domain is an IgG4 Fc domain comprising an amino acid substitution atposition 5228 (Kabat numbering), particularly the amino acidsubstitution S228P. In a more specific aspect, the Fc domain is an IgG4Fc domain comprising amino acid substitutions L235E and S228P and P329G(EU numbering). Such IgG4 Fc domain mutants and their Fcy receptorbinding properties are also described in WO 2012/130831.

Mutant Fc domains can be prepared by amino acid deletion, substitution,insertion or modification using genetic or chemical methods well knownin the art. Genetic methods may include site-specific mutagenesis of theencoding DNA sequence, PCR, gene synthesis, and the like. The correctnucleotide changes can be verified for example by sequencing.

Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBlAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. A suitable such binding assay isdescribed herein. Alternatively, binding affinity of Fc domains or cellactivating bispecific antigen binding molecules comprising an Fc domainfor Fc receptors may be evaluated using cell lines known to expressparticular Fc receptors, such as human NK cells expressing FcγIIIareceptor.

Effector function of an Fc domain, or bispecific antibodies of theinvention comprising an

Fc domain, can be measured by methods known in the art. A suitable assayfor measuring ADCC is described herein. Other examples of in vitroassays to assess ADCC activity of a molecule of interest are describedin U.S. Pat. No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83,7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82,1499-1502 (1985); U.S. Pat. No. 5,821,337; Bruggemann et al., J Exp Med166, 1351-1361 (1987). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTITM non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g. in a animal model such as that disclosed in Clynes et al.,Proc Natl Acad Sci USA 95, 652-656 (1998).

In some embodiments, binding of the Fc domain to a complement component,specifically to C1q, is reduced. Accordingly, in some embodimentswherein the Fc domain is engineered to have reduced effector function,said reduced effector function includes reduced CDC. C1q binding assaysmay be carried out to determine whether the bispecific antibodies of theinvention is able to bind C1q and hence has CDC activity. See e.g., C1qand C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assesscomplement activation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al.,Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743(2004)).

In a particular aspect, the Fc domain comprises a modification promotingthe association of the first and second subunit of the Fc domain.

Fc Domain Modifications Promoting Heterodimerization

The Fc domain comprises different moieties, fused to one or the other ofthe two subunits of the Fc domain that are typically comprised in twonon-identical polypetide chains (“heavy chains”). Recombinantco-expression of these polypeptides and subsequent dimerization leads toseveral possible combinations of the two polypeptides. To improve theyield and purity of the TNF family ligand trimer-containing antigenbinding molecules in recombinant production, it will thus beadvantageous to introduce in the Fc domain of the TNF family ligandtrimer-containing antigen binding molecules of the invention amodification promoting the association of the desired polypeptides.

Accordingly, the Fc domain of the TNF family ligand trimer-containingantigen binding molecules of the invention comprises a modificationpromoting the association of the first and the second subunit of the Fcdomain. The site of most extensive protein-protein interaction betweenthe two subunits of a human IgG Fc domain is in the CH3 domain of the Fcdomain. Thus, said modification is particularly in the CH3 domain of theFc domain.

In a specific aspect, said modification is a so-called “knob-into-hole”modification, comprising a “knob” modification in one of the twosubunits of the Fc domain and a “hole” modification in the other one ofthe two subunits of the Fc domain. Thus, in a particular aspect, theinvention relates to a TNF family ligand trimer-containing antigenbinding molecule as described herein before which comprises an IgGmolecule, wherein the Fc part of the first heavy chain comprises a firstdimerization module and the Fc part of the second heavy chain comprisesa second dimerization module allowing a heterodimerization of the twoheavy chains of the IgG molecule and the first dimerization modulecomprises knobs and the second dimerization module comprises holesaccording to the knob into hole technology.

The knob-into-hole technology is described e.g. in U.S. Pat. No.5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621(1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, themethod involves introducing a protuberance (“knob”) at the interface ofa first polypeptide and a corresponding cavity (“hole”) in the interfaceof a second polypeptide, such that the protuberance can be positioned inthe cavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine).

Accordingly, in a particular aspect, in the CH3 domain of the firstsubunit of the Fc domain of the TNF family ligand trimer-containingantigen binding molecules of the invention an amino acid residue isreplaced with an amino acid residue having a larger side chain volume,thereby generating a protuberance within the CH3 domain of the firstsubunit which is positionable in a cavity within the CH3 domain of thesecond subunit, and in the CH3 domain of the second subunit of the Fcdomain an amino acid residue is replaced with an amino acid residuehaving a smaller side chain volume, thereby generating a cavity withinthe CH3 domain of the second subunit within which the protuberancewithin the CH3 domain of the first subunit is positionable.

The protuberance and cavity can be made by altering the nucleic acidencoding the polypeptides, e.g. by site-specific mutagenesis, or bypeptide synthesis.

In a specific aspect, in the CH3 domain of the first subunit of the Fcdomain the threonine residue at position 366 is replaced with atryptophan residue (T366W), and in the CH3 domain of the second subunitof the Fc domain the tyrosine residue at position 407 is replaced with avaline residue (Y407V). More particularly, in the second subunit of theFc domain additionally the threonine residue at position 366 is replacedwith a serine residue (T366S) and the leucine residue at position 368 isreplaced with an alanine residue (L368A). More particularly, in thefirst subunit of the Fc domain additionally the serine residue atposition 354 is replaced with a cysteine residue (S354C), and in thesecond subunit of the Fc domain additionally the tyrosine residue atposition 349 is replaced by a cysteine residue (Y349C). The introductionof these two cysteine residues results in the formation of a disulfidebridge between the two subunits of the Fc domain. The disulfide bridgefurther stabilizes the dimer (Carter, J Immunol Methods 248, 7-15(2001)).

In an alternative aspect, a modification promoting association of thefirst and the second subunit of the Fc domain comprises a modificationmediating electrostatic steering effects, e.g. as described in PCTpublication WO 2009/089004. Generally, this method involves replacementof one or more amino acid residues at the interface of the two Fc domainsubunits by charged amino acid residues so that homodimer formationbecomes electrostatically unfavorable but heterodimerizationelectrostatically favorable.

Particular TNF Family Ligand Trimer-Containing Antigen Binding Molecules

In another aspect, provided is a monovalent TNF family ligandtrimer-containing antigen binding molecule as described herein before,comprising one moiety capable of specific binding to a target cellantigen.

In particular, the TNF family ligand trimer-containing antigen bindingmolecule of the invention comprising (c) an Fc domain composed of afirst and a second subunit capable of stable association furthercomprises (a) a Fab molecule capable of specific binding to a targetcell antigen, wherein the Fab heavy chain is fused at the C-terminus tothe N-terminus of a CH2 domain in the Fc domain.

Furthermore, the invention provides a TNF family ligandtrimer-containing antigen binding molecule as described herein before,wherein the antigen binding molecule comprises

-   (a) a heavy chain and a light chain, both comprising a Fab molecule    capable of specific binding to a target cell antigen, and-   (b) a fusion protein comprising the amino acid sequence of SEQ ID    NO:15.

In a further aspect, the TNF family ligand trimer-containing antigenbinding molecule comprises

-   (i) a first heavy chain comprising the VH domain of a Fab molecule    capable of specific binding to a target cell antigen,-   (ii) a light chain comprising the VL domain of a Fab molecule    capable of specific binding to a target cell antigen, and-   (iii) a second heavy chain comprising the amino acid sequence of SEQ    ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8.

In a particular aspect, the TNF family ligand trimer-containing antigenbinding molecule comprises (i) a first heavy chain comprising a VHdomain comprising an amino acid sequence of SEQ ID NO:16 or a VH domaincomprising an amino acid sequence of SEQ ID NO:110, (ii) a light chaincomprising a VL domain comprising an amino acid sequence of SEQ ID NO:17or a VL domain comprising an amino acid sequence of SEQ ID NO:111, and(iii) a second heavy chain comprising the amino acid sequence of SEQ IDNO:5 or SEQ ID NO:103 or SEQ ID NO:8. In another aspect, TNF familyligand trimer-containing antigen binding molecule comprises (i) a firstheavy chain comprising a VH domain comprising an amino acid sequence ofSEQ ID NO:118, (ii) a light chain comprising a VL domain comprising anamino acid sequence of SEQ ID NO:119, and (iii) a second heavy chaincomprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:103 orSEQ ID NO:8. In a further aspect, the TNF family ligandtrimer-containing antigen binding molecule comprises (i) a first heavychain comprising a VH domain comprising an amino acid sequence of SEQ IDNO:126 or a VH domain comprising an amino acid sequence of SEQ IDNO:134, (ii) a light chain comprising a VL domain comprising an aminoacid sequence of SEQ ID NO:127 or a VL domain comprising an amino acidsequence of SEQ ID NO:135, and (iii) a second heavy chain comprising theamino acid sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8.

In another aspect, provided is a bivalent TNF family ligandtrimer-containing antigen binding molecule, comprising two moietiescapable of specific binding to a target cell antigen.

In one aspect, the TNF family ligand trimer-containing antigen bindingmolecule comprises

-   (i) a first heavy chain comprising a VH domain or a VL domain of a    Fab molecule capable of specific binding to a target cell antigen,-   (ii) two light chains comprising each a VL domain or a VH domain of    a Fab molecule capable of specific binding to a target cell antigen,    and-   (iii) a second heavy chain comprising a VH domain or a VL domain of    a Fab molecule capable of specific binding to a target cell antigen    and the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ    ID NO:8.

In one aspect, the TNF family ligand trimer-containing antigen bindingmolecule comprises

-   (i) a first heavy chain comprising a VH domain of a Fab molecule    capable of specific binding to a target cell antigen,-   (ii) two light chains comprising each the VL domain of a Fab    molecule capable of specific binding to a target cell antigen, and-   (iii) a second heavy chain comprising a VH domain of a Fab molecule    capable of specific binding to a target cell antigen and the amino    acid sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8.

In a particular aspect, the TNF family ligand trimer-containing antigenbinding molecule comprises (i) a first heavy chain comprising a VHdomain comprising an amino acid sequence of SEQ ID NO:16 or a VH domaincomprising an amino acid sequence of SEQ ID NO:110, (ii) two lightchains comprising each a VL domain comprising an amino acid sequence ofSEQ ID NO:17 or a VL domain comprising an amino acid sequence of SEQ IDNO:111, and (iii) a second heavy chain comprising a VH domain comprisingan amino acid sequence of SEQ ID NO:16 or a VH domain comprising anamino acid sequence of SEQ ID NO:110 and the amino acid sequence of SEQID NO:5 or SEQ ID NO:103 or SEQ ID NO:8. In another aspect, the TNFfamily ligand trimer-containing antigen binding molecule comprises (i) afirst heavy chain comprising a VH domain comprising an amino acidsequence of SEQ ID NO:118, (ii) two light chains comprising each a VLdomain comprising an amino acid sequence of SEQ ID NO:119, and (iii) asecond heavy chain comprising a VH domain comprising an amino acidsequence of SEQ ID NO:118 and the amino acid sequence of SEQ ID NO:5 orSEQ ID NO:103 or SEQ ID NO:8. In yet another aspect, the TNF familyligand trimer-containing antigen binding molecule comprises (i) a firstheavy chain comprising a VH domain comprising an amino acid sequence ofSEQ ID NO:126 or a VH domain comprising an amino acid sequence of SEQ IDNO:134, (ii) two light chains comprising a VL domain comprising an aminoacid sequence of SEQ ID NO:127 or a VL domain comprising an amino acidsequence of SEQ ID NO:135, and (iii) a second heavy chain comprising aVH domain comprising an amino acid sequence of SEQ ID NO:126 or a VHdomain comprising an amino acid sequence of SEQ ID NO:134 and the aminoacid sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8.

In one aspect, provided is a TNF family ligand trimer-containing antigenbinding molecule according to the invention, wherein the antigen bindingmolecule comprises

-   (a) a heavy chain comprising a VH domain comprising (i) CDR-H1    comprising the amino acid sequence of SEQ ID NO:9, (ii) CDR-H2    comprising the amino acid sequence of SEQ ID NO:10 and (iii) CDR-H3    comprising the amino acid sequence of SEQ ID NO:11, and a light    chain comprising a VL domain comprising (iv) CDR-L1 comprising the    amino acid sequence of SEQ ID NO:12, (v) CDR-L2 comprising the amino    acid sequence of SEQ ID NO:13 and (vi) CDR-L3 comprising the amino    acid sequence of SEQ ID NO:14, and-   (b) a fusion protein comprising the amino acid sequence of SEQ ID    NO:15.

In a further aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule according to the invention,wherein the antigen binding molecule comprises

-   (a) a moiety capable of specific binding to FAP comprising a VH    domain comprising the amino acid sequence of SEQ ID NO:16 and a VL    domain comprising the amino acid sequence of SEQ ID NO:17 and-   (b) a fusion protein comprising the amino acid sequence of SEQ ID    NO:15.

In a particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as described herein before,wherein the antigen binding molecule comprises

-   (i) a variable heavy chain comprising the amino acid sequence of SEQ    ID NO:16,-   (ii) a variable light chain comprising the amino acid sequence of    SEQ ID NO:17, and-   (iii) a polypeptide comprising the amino acid sequence of SEQ ID    NO:5.

In a particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as described herein before,wherein the antigen binding molecule comprises

-   (i) a heavy chain comprising the amino acid sequence of SEQ ID    NO:16,-   (ii) a light chain comprising the amino acid sequence of SEQ ID    NO:17, and-   (iii) a fusion protein comprising the amino acid sequence of SEQ ID    NO:15.

In another aspect, provided is a TNF family ligand trimer-containingantigen binding molecule as described hereinbefore, wherein the antigenbinding molecule comprises

-   (a) a heavy chain and a light chain, both comprising a Fab molecule    capable of specific binding to a target cell antigen, and-   (b) the fusion protein comprising the amino acid sequence of SEQ ID    NO:20.

In one aspect, provided is a TNF family ligand trimer-containing antigenbinding molecule according to the invention, wherein the antigen bindingmolecule comprises

-   (a) a heavy chain comprising a VH domain comprising (i) CDR-H1    comprising the amino acid sequence of SEQ ID NO:9, (ii) CDR-H2    comprising the amino acid sequence of SEQ ID NO:10 and (iii) CDR-H3    comprising the amino acid sequence of SEQ ID NO:11, and a light    chain comprising a VL domain comprising (iv) CDR-L1 comprising the    amino acid sequence of SEQ ID NO:12, (v) CDR-L2 comprising the amino    acid sequence of SEQ ID NO:13 and (vi) CDR-L3 comprising the amino    acid sequence of SEQ ID NO:14, and-   (b) a fusion protein comprising the amino acid sequence of SEQ ID    NO:20.

In a further aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule according to the invention,wherein the antigen binding molecule comprises

-   (a) a moiety capable of specific binding to FAP comprising a VH    domain comprising the amino acid sequence of SEQ ID NO:16 and a VL    domain comprising the amino acid sequence of SEQ ID NO:17 and-   (b) a fusion protein comprising the amino acid sequence of SEQ ID    NO:20.

In a particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as described herein before,wherein the antigen binding molecule comprises

-   (i) a variable heavy chain comprising the amino acid sequence of SEQ    ID NO:16,-   (ii) a variable light chain comprising the amino acid sequence of    SEQ ID NO:17, and-   (iii) a polypeptide comprising the amino acid sequence of SEQ ID    NO:8.

In a particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as described herein before,wherein the antigen binding molecule comprises

-   (i) a heavy chain comprising the amino acid sequence of SEQ ID    NO:16,-   (ii) a light chain comprising the amino acid sequence of SEQ ID    NO:17, and-   (iii) a fusion protein comprising the amino acid sequence of SEQ ID    NO:20.

TNF Family Ligand Trimer-Containing Antigen Binding Molecules, Whereinthe Target Cell Antigen is FAP

In a particular aspect, the target cell antigen is Fibroblast ActivationProtein (FAP). In one aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule as decribed herein before,wherein the moiety capable of specific binding to FAP comprises

-   (a) a VH domain comprising (i) CDR-H1 comprising the amino acid    sequence of SEQ ID NO:9, (ii) CDR-H2 comprising the amino acid    sequence of SEQ ID NO:10 and (iii) CDR-H3 comprising the amino acid    sequence of SEQ ID NO:11, and a VL domain comprising (iv) CDR-L1    comprising the amino acid sequence of SEQ ID NO:12, (v) CDR-L2    comprising the amino acid sequence of SEQ ID NO:13 and (vi) CDR-L3    comprising the amino acid sequence of SEQ ID NO:14, or-   (b) a VH domain comprising (i) CDR-H1 comprising the amino acid    sequence of SEQ ID NO:104, (ii) CDR-H2 comprising the amino acid    sequence of SEQ ID NO:105 and (iii) CDR-H3 comprising the amino acid    sequence of SEQ ID NO:106, and a VL domain comprising (iv) CDR-L1    comprising the amino acid sequence of SEQ ID NO:107, (v) CDR-L2    comprising the amino acid sequence of SEQ ID NO:108 and (vi) CDR-L3    comprising the amino acid sequence of SEQ ID NO:109.

In one aspect, the moiety capable of specific binding to FAP comprises aVH domain comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:9, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:10 and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:11, and a VL domain comprising (iv) CDR-L1 comprising the amino acidsequence of SEQ ID NO:12, (v) CDR-L2 comprising the amino acid sequenceof

SEQ ID NO:13 and (vi) CDR-L3 comprising the amino acid sequence of SEQID NO:14. In another particular aspect, the moiety capable of specificbinding to FAP comprises a VH domain comprising (i) CDR-H1 comprisingthe amino acid sequence of SEQ ID NO:104, (ii) CDR-H2 comprising theamino acid sequence of SEQ ID NO:105 and (iii) CDR-H3 comprising theamino acid sequence of SEQ ID NO:106, and a VL domain comprising (iv)CDR-L1 comprising the amino acid sequence of SEQ ID NO:107, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:108 and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:109.

In a further aspect, the moiety capable of specific binding to FAPcomprises a heavy chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:16 and a light chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO:17. In a particular aspect, the moiety capable of specificbinding to FAP comprises a heavy chain variable region comprising theamino acid sequence of SEQ ID NO:16 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:17. In a specificaspect, the moiety capable of specific binding to FAP comprises a VHdomain consisting of amino acid sequence of SEQ ID NO:16 and a VL domainconsisting of the amino acid sequence of SEQ ID NO:17.

In a further particular aspect, the TNF family ligand trimer-containingantigen binding molecule of the invention comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:18 and a light chaincomprising the amino acid sequence of SEQ ID NO:19. In a specificaspect, the TNF family ligand trimer-containing antigen binding moleculecomprises a heavy chain consisting of amino acid sequence of SEQ IDNO:18 and a light chain consisting of the amino acid sequence of SEQ IDNO:19.

In another aspect, the moiety capable of specific binding to FAPcomprises a heavy chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:110 and a light chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO:111. In a particular aspect, the moiety capable of specificbinding to FAP comprises a heavy chain variable region comprising theamino acid sequence of SEQ ID NO:110 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:111. In a specificaspect, the moiety capable of specific binding to FAP comprises a VHdomain consisting of amino acid sequence of SEQ ID NO:110 and a VLdomain consisting of the amino acid sequence of SEQ ID NO:111.

In one aspect, provided is a monovalent TNF family ligandtrimer-containing antigen binding molecule as described herein before,comprising one moiety capable of specific binding to FAP.

In a particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as described herein before,wherein the antigen binding molecule comprises

-   (i) a variable heavy chain comprising the amino acid sequence of SEQ    ID NO:18,-   (ii) a variable light chain comprising the amino acid sequence of    SEQ ID NO:19, and-   (iii) a fusion protein comprising the amino acid sequence of SEQ ID    NO:15.

In a further particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as described herein before,wherein the antigen binding molecule comprises

-   (i) a variable heavy chain comprising the amino acid sequence of SEQ    ID NO:140,-   (ii) a variable light chain comprising the amino acid sequence of    SEQ ID NO:142, and-   (iii) a fusion protein comprising the amino acid sequence of SEQ ID    NO:15.

In another particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as described herein before,wherein the antigen binding molecule comprises

-   (i) a variable heavy chain comprising the amino acid sequence of SEQ    ID NO:18,-   (ii) a variable light chain comprising the amino acid sequence of    SEQ ID NO:19, and-   (iii) a fusion protein comprising the amino acid sequence of SEQ ID    NO:20.

In another aspect, provided is a bivalent TNF family ligandtrimer-containing antigen binding molecule as described herein before,comprising two moieties capable of specific binding to FAP.

In a particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as described herein before,wherein the antigen binding molecule comprises

-   (i) a variable heavy chain comprising the amino acid sequence of SEQ    ID NO:140,-   (ii) a variable light chain comprising the amino acid sequence of    SEQ ID NO:142, and-   (iii) a fusion protein comprising the amino acid sequence of SEQ ID    NO:141.

TNF Family Ligand Trimer-Containing Antigen Binding Molecules, Whereinthe Target Cell Antigen is CEA

In another aspect, the target cell antigen is CEA. In one aspect, theinvention provides a TNF family ligand trimer-containing antigen bindingmolecule as decribed herein before, wherein the moiety capable ofspecific binding to CEA comprises a VH domain comprising (i) CDR-H1comprising the amino acid sequence of SEQ ID NO:112, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:113 and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:114, and a VL domaincomprising (iv) CDR-L1 comprising the amino acid sequence of SEQ IDNO:115, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:116and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:117.

In a further aspect, the moiety capable of specific binding to CEAcomprises a heavy chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:118 and a light chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO:119. In a particular aspect, the moiety capable of specificbinding to CEA comprises a heavy chain variable region comprising theamino acid sequence of SEQ ID NO:118 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:119. In a specificaspect, the moiety capable of specific binding to FAP comprises a VHdomain consisting of amino acid sequence of SEQ ID NO:118 and a VLdomain consisting of the amino acid sequence of SEQ ID NO:119.

In one aspect, provided is a monovalent TNF family ligandtrimer-containing antigen binding molecule as described herein before,comprising one moiety capable of specific binding to CEA.

In a particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as described herein before,wherein the antigen binding molecule comprises

-   (i) a variable heavy chain comprising the amino acid sequence of SEQ    ID NO:146,-   (ii) a variable light chain comprising the amino acid sequence of    SEQ ID NO:148, and-   (iii) a fusion protein comprising the amino acid sequence of SEQ ID    NO:15.

In another aspect, provided is a bivalent TNF family ligandtrimer-containing antigen binding molecule as described herein before,comprising two moieties capable of specific binding to CEA.

In a particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as described herein before,wherein the antigen binding molecule comprises

-   (i) a variable heavy chain comprising the amino acid sequence of SEQ    ID NO:146,-   (ii) a variable light chain comprising the amino acid sequence of    SEQ ID NO:148, and (iii) a fusion protein comprising the amino acid    sequence of SEQ ID NO:147.

TNF Family Ligand Trimer-Containing Antigen Binding Molecules, Whereinthe Target Cell Antigen is CD19

In yet another aspect, the target cell antigen is CD19. In one aspect,the invention provides a TNF family ligand trimer-containing antigenbinding molecule as decribed herein before, wherein the moiety capableof specific binding to CD19 comprises (a) a VH domain comprising (i)CDR-H1 comprising the amino acid sequence of SEQ ID NO:120, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:121 and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:122, and a VL domaincomprising (iv) CDR-L1 comprising the amino acid sequence of SEQ IDNO:123, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:124and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:125, or(b) a VH domain comprising (i) CDR-H1 comprising the amino acid sequenceof SEQ ID NO:128, (ii) CDR-H2 comprising the amino acid sequence of SEQID NO:129 and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:130, and a VL domain comprising (iv) CDR-L1 comprising the amino acidsequence of SEQ ID NO:131, (v) CDR-L2 comprising the amino acid sequenceof SEQ ID NO:132 and (vi) CDR-L3 comprising the amino acid sequence ofSEQ ID NO:133.

In a further aspect, the moiety capable of specific binding to CD19comprises a heavy chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:126 and a light chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO:127. In another aspect, the moiety capable of specific bindingto CD19 comprises a heavy chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:134 and a light chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO:135. In a particular aspect, the moiety capable of specificbinding to CD19 comprises a heavy chain variable region comprising theamino acid sequence of SEQ ID NO:126 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:127. In a specificaspect, the moiety capable of specific binding to FAP comprises a VHdomain consisting of amino acid sequence of SEQ ID NO:118 and a VLdomain consisting of the amino acid sequence of SEQ ID NO:119. Inanother particular aspect, moiety capable of specific binding to CD19comprises a variable heavy chain comprising an amino acid sequence ofSEQ ID NO:134 and a variable light chain comprising an amino acidsequence of SEQ ID NO:135. In a specific aspect, the moiety capable ofspecific binding to FAP comprises a VH domain consisting of amino acidsequence of SEQ ID NO:134 and a VL domain consisting of the amino acidsequence of SEQ ID NO:135.

In one aspect, provided is a monovalent TNF family ligandtrimer-containing antigen binding molecule as described herein before,comprising one moiety capable of specific binding to CD19.

In a particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as described herein before,wherein the antigen binding molecule comprises

-   (i) a variable heavy chain comprising the amino acid sequence of SEQ    ID NO:152,-   (ii) a variable light chain comprising the amino acid sequence of    SEQ ID NO:154, and-   (iii) a fusion protein comprising the amino acid sequence of SEQ ID    NO:15.

In a further particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as described herein before,wherein the antigen binding molecule comprises

-   (i) a variable heavy chain comprising the amino acid sequence of SEQ    ID NO:158,-   (ii) a variable light chain comprising the amino acid sequence of    SEQ ID NO:160, and-   (iii) a fusion protein comprising the amino acid sequence of SEQ ID    NO:15.

In another aspect, provided is a bivalent TNF family ligandtrimer-containing antigen binding molecule as described herein before,comprising two moieties capable of specific binding to CD19.

In a particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as described herein before,wherein the antigen binding molecule comprises

-   (i) a variable heavy chain comprising the amino acid sequence of SEQ    ID NO:152,-   (ii) a variable light chain comprising the amino acid sequence of    SEQ ID NO:154, and-   (iii) a fusion protein comprising the amino acid sequence of SEQ ID    NO:153.

In a further particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as described herein before,wherein the antigen binding molecule comprises

-   (i) a variable heavy chain comprising the amino acid sequence of SEQ    ID NO:158,-   (ii) a variable light chain comprising the amino acid sequence of    SEQ ID NO:160, and-   (iii) a fusion protein comprising the amino acid sequence of SEQ ID    NO:159.

Polynucleotides

The invention further provides isolated polynucleotides encoding a TNFfamily ligand trimer-containing antigen binding molecule as describedherein or a fragment thereof.

The isolated polynucleotides encoding TNF ligand trimer-containingantigen binding molecules of the invention may be expressed as a singlepolynucleotide that encodes the entire antigen binding molecule or asmultiple (e.g., two or more) polynucleotides that are co-expressed.Polypeptides encoded by polynucleotides that are co-expressed mayassociate through, e.g., disulfide bonds or other means to form afunctional antigen binding molecule. For example, the light chainportion of an immunoglobulin may be encoded by a separate polynucleotidefrom the heavy chain portion of the immunoglobulin. When co-expressed,the heavy chain polypeptides will associate with the light chainpolypeptides to form the immunoglobulin.

In some aspects, the isolated polynucleotide encodes the entire TNFfamily ligand trimer-containing antigen binding molecule according tothe invention as described herein. In other embodiments, the isolatedpolynucleotide encodes a polypeptide comprised in the TNF family ligandtrimer-containing antigen binding molecule according to the invention asdescribed herein.

In one aspect, the present invention is directed to an isolatedpolynucleotide encoding a TNF family ligand trimer-containing antigenbinding molecule, wherein the polynucleotide comprises (a) a sequencethat encodes a moiety capable of specific binding to a target cellantigen, (b) a sequence that encodes polypeptide comprising threeectodomains of a TNF ligand family member or fragments thereof that areconnected to each other by peptide linkers and (c) a sequence thatencodes a Fc domain composed of a first and a second subunit capable ofstable association.

In another aspect, provided is an isolated polynucleotide encoding a4-1BB ligand trimer-containing antigen binding molecule, wherein thepolynucleotide comprises (a) a sequence that encodes a moiety capable ofspecific binding to a target cell antigen, (b) a sequence that encodespolypeptide comprising three ectodomains of 4-1BBL or fragments thereofthat are connected to each other by peptide linkers and (c) a sequencethat encodes a Fc domain composed of a first and a second subunitcapable of stable association.

In a further aspect, the invention is directed to an isolatedpolynucleotide comprising a sequence that encodes a polypeptidecomprising three 4-1BBL fragments comprising an amino acid sequence thatis at least about 90%, 95%, 98% or 100% identical to an amino acidsequence shown in SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO:101 and SEQ ID NO:102. In oneaspect, provided is an isolated polynucleotide comprising a sequencethat encodes a polypeptide comprising the amino acid sequence of SEQ IDNO:5 or SEQ ID NO:103.

In another aspect, provided is an isolated polynucleotide encoding aOX40 ligand trimer-containing antigen binding molecule, wherein thepolynucleotide comprises (a) a sequence that encodes a moiety capable ofspecific binding to a target cell antigen, (b) a sequence that encodespolypeptide comprising three ectodomains of OX40L or fragments thereofthat are connected to each other by peptide linkers and (c) a sequencethat encodes a Fc domain composed of a first and a second subunitcapable of stable association.

In a further aspect, the invention is directed to an isolatedpolynucleotide comprising a sequence that encodes a polypeptidecomprising three OX40L fragments comprising an amino acid sequence thatis at least about 90%, 95%, 98% or 100% identical to an amino acidsequence shown in SEQ ID NO:6 or SEQ ID NO:7. In one aspect, provided isan isolated polynucleotide comprising a sequence that encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:8.

In another aspect, provided is an isolated polynucleotide encoding a TNFfamily ligand trimer-containing antigen binding molecule, wherein thepolynucleotide comprises (a) a sequence that encodes a moiety capable ofspecific binding to a target cell antigen, (b) a sequence that encodes apolypeptide comprising three ectodomains of a TNF ligand family memberor fragments thereof that are connected to each other by peptide linkersand that is fused at the C-terminal amino acid to the N-terminal aminoacid of a CH2 domain in the Fc domain (c) a sequence that encodes a Fcdomain composed of a first and a second subunit capable of stableassociation. In a further aspect, provided is an isolated polynucleotideencoding a TNF family ligand trimer-containing antigen binding molecule,wherein the polynucleotide comprises (a) a sequence that encodes amoiety capable of specific binding to a target cell antigen, (b) asequence that encodes a polypeptide comprising three ectodomains of aTNF ligand family member or fragments thereof that are connected to eachother by peptide linkers is fused at the N-terminal amino acid to theC-terminal amino acid of a CH3 domain in the Fc domain and (c) asequence that encodes a Fc domain composed of a first and a secondsubunit capable of stable association.

In one aspect, the invention is directed to an isolated polynucleotidethat comprises a sequence that encodes an IgG1 Fc domain. In aparticular aspect, the isolated polynucleotide comprises a sequence thatencodes an Fc domain with amino acid substitutions at positions 234, 235and/or 329 (EU numbering).

In another aspect, the invention is directed to an isolatedpolynucleotide that comprises a sequence that is at least about 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acidsequence as shown in SEQ ID NO:66. In a further aspect, provided is anisolated polynucleotide that comprises a sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acidsequence as shown in SEQ ID NO:87.

In a further aspect, the invention is directed to an isolatedpolynucleotide encoding a TNF family ligand trimer-containing antigenbinding molecule, wherein the polynucleotide comprises sequencesencoding a heavy chain and a light chain, both comprising a Fab moleculecapable of specific binding to a target cell antigen, and the nucleicacid sequence of SEQ ID NO:66. In a further aspect, the invention isdirected to an isolated polynucleotide encoding a TNF family ligandtrimer-containing antigen binding molecule, wherein the polynucleotidecomprises sequences encoding a heavy chain and a light chain, bothcomprising a Fab molecule capable of specific binding to a target cellantigen, and the nucleic acid sequence of SEQ ID NO:87.

In one aspect, provided is an isolated polynucleotide encoding a 4-1BBLtrimer-containing antigen binding molecule, wherein the polynucleotidecomprises sequences encoding (a) a heavy chain comprising a VH domaincomprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:9,(ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:10 and (iii)CDR-H3 comprising the amino acid sequence of SEQ ID NO:11, and a lightchain comprising a

VL domain comprising (iv) CDR-L1 comprising the amino acid sequence ofSEQ ID NO:12, (v) CDR-L2 comprising the amino acid sequence of SEQ IDNO:13 and (vi) CDR-L3 comprising the amino acid sequence of SEQ IDNO:14, and (b) a polypeptide comprising the amino acid sequence of SEQID NO:5.

In another aspect, provided is an isolated polynucleotide encoding a4-1BBL trimer-containing antigen binding molecule, wherein thepolynucleotide comprises sequences encoding (a) a heavy chain comprisinga VH domain comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:104, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:105 and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:106, and a light chain comprising a VL domain comprising (iv) CDR-L1comprising the amino acid sequence of SEQ ID NO:107, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:108 and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:109, and (b) apolypeptide comprising the amino acid sequence of SEQ ID NO:5.

In a further aspect, provided is an isolated polynucleotide encoding a4-1BBL trimer-containing antigen binding molecule, wherein thepolynucleotide comprises sequences encoding (a) a heavy chain comprisinga VH domain comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:112, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:113 and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:114, and a light chain comprising a VL domain comprising (iv) CDR-L1comprising the amino acid sequence of SEQ ID NO:115, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:116 and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:117, and (b) apolypeptide comprising the amino acid sequence of SEQ ID NO:5.

In yet another aspect, provided is an isolated polynucleotide encoding a4-1BBL trimer-containing antigen binding molecule, wherein thepolynucleotide comprises sequences encoding (a) a heavy chain comprisinga VH domain comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:120, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:121 and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:122, and a light chain comprising a VL domain comprising (iv) CDR-L1comprising the amino acid sequence of SEQ ID NO:123, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:124 and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:125, and (b) apolypeptide comprising the amino acid sequence of SEQ ID NO:5.

In a further aspect, provided is an isolated polynucleotide encoding a4-1BBL trimer-containing antigen binding molecule, wherein thepolynucleotide comprises sequences encoding (a) a heavy chain comprisinga VH domain comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:128, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:129 and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:130, and a light chain comprising a VL domain comprising (iv) CDR-L1comprising the amino acid sequence of SEQ ID NO:131, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:132 and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:133, and (b) apolypeptide comprising the amino acid sequence of SEQ ID NO:5.

In a further aspect, the invention provides an isolated polynucleotideencoding a 4-1BBL trimer-containing antigen binding molecule, whereinthe polynucleotide comprises sequences encoding (a) a moiety capable ofspecific binding to FAP comprising a VH domain consisting of amino acidsequence of SEQ ID NO:16 and a VL domain consisting of the amino acidsequence of SEQ ID NO:17 and b) a polypeptide comprising the amino acidsequence of SEQ ID NO:5.

In a particular aspect, the invention provides an isolatedpolynucleotide encoding a 4-1BBL trimer-containing antigen bindingmolecule, wherein the polynucleotide comprises sequences encoding (i) aheavy chain comprising the amino acid sequence of SEQ ID NO:16, (ii) alight chain comprising the amino acid sequence of SEQ ID NO:17, and(iii) a fusion protein comprising the amino acid sequence of SEQ IDNO:15.

In a further aspect, the invention provides an isolated polynucleotideencoding a 4-1BBL trimer-containing antigen binding molecule, whereinthe polynucleotide comprises sequences encoding (a) a moiety capable ofspecific binding to FAP comprising a VH domain consisting of amino acidsequence of SEQ ID NO:110 and a VL domain consisting of the amino acidsequence of SEQ ID NO:111 and b) a polypeptide comprising the amino acidsequence of SEQ ID NO:5.

In a particular aspect, the invention provides an isolatedpolynucleotide encoding a 4-1BBL trimer-containing antigen bindingmolecule, wherein the polynucleotide comprises sequences encoding (i) aheavy chain comprising the amino acid sequence of SEQ ID NO:110, (ii) alight chain comprising the amino acid sequence of SEQ ID NO:111, and(iii) a fusion protein comprising the amino acid sequence of SEQ IDNO:15.

In another aspect, provided is an isolated polynucleotide encoding aOX40L trimer-containing antigen binding molecule, wherein thepolynucleotide comprises sequences encoding (a) a heavy chain comprisinga VH domain comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:9, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:10 and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:11, and a light chain comprising a VL domain comprising (iv) CDR-L1comprising the amino acid sequence of SEQ ID NO:12, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:13 and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:14, and (b) apolypeptide comprising the amino acid sequence of SEQ ID NO:8.

In a further aspect, the invention provides an isolated polynucleotideencoding a OX40L trimer-containing antigen binding molecule, wherein thepolynucleotide comprises sequences encoding (a) a moiety capable ofspecific binding to FAP comprising a VH domain consisting of amino acidsequence of SEQ ID NO:16 and a VL domain consisting of the amino acidsequence of SEQ ID NO:17 and b) a polypeptide comprising the amino acidsequence of SEQ ID NO:8.

In a particular aspect, the invention provides an isolatedpolynucleotide encoding a OX40L trimer-containing antigen bindingmolecule, wherein the polynucleotide comprises sequences encoding (i) aheavy chain comprising the amino acid sequence of SEQ ID NO:16, (ii) alight chain comprising the amino acid sequence of SEQ ID NO:17, and(iii) a fusion protein comprising the amino acid sequence of SEQ IDNO:20.

In certain embodiments the polynucleotide or nucleic acid is DNA. Inother embodiments, a polynucleotide of the present invention is RNA, forexample, in the form of messenger RNA (mRNA). RNA of the presentinvention may be single stranded or double stranded.

Recombinant Methods

TNF family ligand trimer-containing antigen binding molecules of theinvention may be obtained, for example, by solid-state peptide synthesis(e.g. Merrifield solid phase synthesis) or recombinant production. Forrecombinant production one or more polynucleotide encoding the TNFfamily ligand trimer-containing antigen binding molecule or polypeptidefragments thereof, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such polynucleotide may be readily isolated and sequenced usingconventional procedures. In one aspect of the invention, a vector,preferably an expression vector, comprising one or more of thepolynucleotides of the invention is provided. Methods which are wellknown to those skilled in the art can be used to construct expressionvectors containing the coding sequence of the TNF family ligandtrimer-containing antigen binding molecule (fragment) along withappropriate transcriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination/genetic recombination. See, for example, thetechniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORYMANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates andWiley Interscience, N.Y. (1989). The expression vector can be part of aplasmid, virus, or may be a nucleic acid fragment. The expression vectorincludes an expression cassette into which the polynucleotide encodingthe TNF family ligand trimer-containing antigen binding molecule orpolypeptide fragments thereof (i.e. the coding region) is cloned inoperable association with a promoter and/or other transcription ortranslation control elements. As used herein, a “coding region” is aportion of nucleic acid which consists of codons translated into aminoacids. Although a “stop codon” (TAG, TGA, or TAA) is not translated intoan amino acid, it may be considered to be part of a coding region, ifpresent, but any flanking sequences, for example promoters, ribosomebinding sites, transcriptional terminators, introns, 5′ and 3′untranslated regions, and the like, are not part of a coding region. Twoor more coding regions can be present in a single polynucleotideconstruct, e.g. on a single vector, or in separate polynucleotideconstructs, e.g. on separate (different) vectors. Furthermore, anyvector may contain a single coding region, or may comprise two or morecoding regions, e.g. a vector of the present invention may encode one ormore polypeptides, which are post- or co-translationally separated intothe final proteins via proteolytic cleavage. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a polynucleotide encoding theTNF family ligand trimer-containing antigen binding molecule of theinvention or polypeptide fragments thereof, or variants or derivativesthereof. Heterologous coding regions include without limitationspecialized elements or motifs, such as a secretory signal peptide or aheterologous functional domain. An operable association is when a codingregion for a gene product, e.g. a polypeptide, is associated with one ormore regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operably associated” if inductionof promoter function results in the transcription of mRNA encoding thedesired gene product and if the nature of the linkage between the twoDNA fragments does not interfere with the ability of the expressionregulatory sequences to direct the expression of the gene product orinterfere with the ability of the DNA template to be transcribed. Thus,a promoter region would be operably associated with a nucleic acidencoding a polypeptide if the promoter was capable of effectingtranscription of that nucleic acid. The promoter may be a cell-specificpromoter that directs substantial transcription of the DNA only inpredetermined cells. Other transcription control elements, besides apromoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription.

Suitable promoters and other transcription control regions are disclosedherein. A variety of transcription control regions are known to thoseskilled in the art. These include, without limitation, transcriptioncontrol regions, which function in vertebrate cells, such as, but notlimited to, promoter and enhancer segments from cytomegaloviruses (e.g.the immediate early promoter, in conjunction with intron-A), simianvirus 40 (e.g. the early promoter), and retroviruses (such as, e.g. Roussarcoma virus). Other transcription control regions include thosederived from vertebrate genes such as actin, heat shock protein, bovinegrowth hormone and rabbit {circumflex over (α)}-globin, as well as othersequences capable of controlling gene expression in eukaryotic cells.Additional suitable transcription control regions includetissue-specific promoters and enhancers as well as inducible promoters(e.g. promoters inducible tetracyclins). Similarly, a variety oftranslation control elements are known to those of ordinary skill in theart. These include, but are not limited to ribosome binding sites,translation initiation and termination codons, and elements derived fromviral systems (particularly an internal ribosome entry site, or IRES,also referred to as a CITE sequence). The expression cassette may alsoinclude other features such as an origin of replication, and/orchromosome integration elements such as retroviral long terminal repeats(LTRs), or adeno-associated viral (AAV) inverted terminal repeats(ITRs).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. For example, if secretionof the TNF family ligand trimer-containing antigen binding molecule orpolypeptide fragments thereof is desired, DNA encoding a signal sequencemay be placed upstream of the nucleic acid encoding a TNF family ligandtrimer-containing antigen binding molecule of the invention orpolypeptide fragments thereof. According to the signal hypothesis,proteins secreted by mammalian cells have a signal peptide or secretoryleader sequence which is cleaved from the mature protein once export ofthe growing protein chain across the rough endoplasmic reticulum hasbeen initiated. Those of ordinary skill in the art are aware thatpolypeptides secreted by vertebrate cells generally have a signalpeptide fused to the N-terminus of the polypeptide, which is cleavedfrom the translated polypeptide to produce a secreted or “mature” formof the polypeptide. In certain embodiments, the native signal peptide,e.g. an immunoglobulin heavy chain or light chain signal peptide isused, or a functional derivative of that sequence that retains theability to direct the secretion of the polypeptide that is operablyassociated with it. Alternatively, a heterologous mammalian signalpeptide, or a functional derivative thereof, may be used. For example,the wild-type leader sequence may be substituted with the leadersequence of human tissue plasminogen activator (TPA) or mouseβ-glucuronidase.

DNA encoding a short protein sequence that could be used to facilitatelater purification (e.g. a histidine tag) or assist in labeling thefusion protein may be included within or at the ends of thepolynucleotide encoding a TNF family ligand trimer-containing antigenbinding molecule of the invention or polypeptide fragments thereof.

In a further aspect of the invention, a host cell comprising one or morepolynucleotides of the invention is provided. In certain embodiments ahost cell comprising one or more vectors of the invention is provided.The polynucleotides and vectors may incorporate any of the features,singly or in combination, described herein in relation topolynucleotides and vectors, respectively. In one aspect, a host cellcomprises (e.g. has been transformed or transfected with) a vectorcomprising a polynucleotide that encodes (part of) a TNF family ligandtrimer-containing antigen binding molecule of the invention of theinvention. As used herein, the term “host cell” refers to any kind ofcellular system which can be engineered to generate the fusion proteinsof the invention or fragments thereof. Host cells suitable forreplicating and for supporting expression of antigen binding moleculesare well known in the art. Such cells may be transfected or transducedas appropriate with the particular expression vector and largequantities of vector containing cells can be grown for seeding largescale fermenters to obtain sufficient quantities of the antigen bindingmolecule for clinical applications. Suitable host cells includeprokaryotic microorganisms, such as E. coli, or various eukaryoticcells, such as Chinese hamster ovary cells (CHO), insect cells, or thelike. For example, polypeptides may be produced in bacteria inparticular when glycosylation is not needed. After expression, thepolypeptide may be isolated from the bacterial cell paste in a solublefraction and can be further purified. In addition to prokaryotes,eukaryotic microbes such as filamentous fungi or yeast are suitablecloning or expression hosts for polypeptide-encoding vectors, includingfungi and yeast strains whose glycosylation pathways have been“humanized”, resulting in the production of a polypeptide with apartially or fully human glycosylation pattern. See Gerngross, NatBiotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215(2006).

Suitable host cells for the expression of (glycosylated) polypeptidesare also derived from multicellular organisms (invertebrates andvertebrates). Examples of invertebrate cells include plant and insectcells. Numerous baculoviral strains have been identified which may beused in conjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells. Plant cell cultures can also be utilized ashosts. See e.g. U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548,7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology forproducing antibodies in transgenic plants). Vertebrate cells may also beused as hosts. For example, mammalian cell lines that are adapted togrow in suspension may be useful. Other examples of useful mammalianhost cell lines are monkey kidney CV1 line transformed by SV40 (COS-7);human embryonic kidney line (293 or 293T cells as described, e.g., inGraham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells(BHK), mouse sertoli cells (TM4 cells as described, e.g., in Mather,Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), Africangreen monkey kidney cells (VERO-76), human cervical carcinoma cells(HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A),human lung cells (W138), human liver cells (Hep G2), mouse mammary tumorcells (MMT 060562), TRI cells (as described, e.g., in Mather et al.,Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells.Other useful mammalian host cell lines include Chinese hamster ovary(CHO) cells, including dhfr-CHO cells (Urlaub et al., Proc Natl Acad SciUSA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63 andSp2/0. For a review of certain mammalian host cell lines suitable forprotein production, see, e.g., Yazaki and Wu, Methods in MolecularBiology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.), pp.255-268 (2003). Host cells include cultured cells, e.g., mammaliancultured cells, yeast cells, insect cells, bacterial cells and plantcells, to name only a few, but also cells comprised within a transgenicanimal, transgenic plant or cultured plant or animal tissue. In oneembodiment, the host cell is a eukaryotic cell, preferably a mammaliancell, such as a Chinese Hamster Ovary (CHO) cell, a human embryonickidney (HEK) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).Standard technologies are known in the art to express foreign genes inthese systems. Cells expressing a polypeptide comprising either theheavy or the light chain of an immunoglobulin, may be engineered so asto also express the other of the immunoglobulin chains such that theexpressed product is an immunoglobulin that has both a heavy and a lightchain.

In one aspect, a method of producing a TNF family ligandtrimer-containing antigen binding molecule of the invention orpolypeptide fragments thereof is provided, wherein the method comprisesculturing a host cell comprising polynucleotides encoding the TNF familyligand trimer-containing antigen binding molecule of the invention orpolypeptide fragments thereof, as provided herein, under conditionssuitable for expression of the TNF family ligand trimer-containingantigen binding molecule of the invention or polypeptide fragmentsthereof, and recovering the TNF family ligand trimer-containing antigenbinding molecule of the invention or polypeptide fragments thereof fromthe host cell (or host cell culture medium).

In the TNF family ligand trimer-containing antigen binding molecule ofthe invention, the components (at least one moiety capable of specificbinding to a target cell antigen, one polypeptide comprising threeectodomains of a TNF ligand family member or fragments thereof and a Fcdomain) are not all genetically fused to each other. The polypeptidesare designed such that its components (three ectodomains of a TNF ligandfamily member or fragments thereof and other components such as CH2 andCH3 of the Fc domain) are fused to each other directly or through alinker sequence. The composition and length of the linker may bedetermined in accordance with methods well known in the art and may betested for efficacy. Examples of linker sequences between differentcomponents of the antigen binding molecules of the invention are foundin the sequences provided herein. Additional sequences may also beincluded to incorporate a cleavage site to separate the individualcomponents of the fusion protein if desired, for example anendopeptidase recognition sequence.

In certain embodiments the moieties capable of specific binding to atarget cell antigen (e.g. Fab fragments) forming part of the antigenbinding molecule comprise at least an immunoglobulin variable regioncapable of binding to an antigen. Variable regions can form part of andbe derived from naturally or non-naturally occurring antibodies andfragments thereof. Methods to produce polyclonal antibodies andmonoclonal antibodies are well known in the art (see e.g. Harlow andLane, “Antibodies, a laboratory manual”, Cold Spring Harbor Laboratory,1988). Non-naturally occurring antibodies can be constructed using solidphase-peptide synthesis, can be produced recombinantly (e.g. asdescribed in U.S. Pat. No. 4,186,567) or can be obtained, for example,by screening combinatorial libraries comprising variable heavy chainsand variable light chains (see e.g. U.S. Pat. No. 5,969,108 toMcCafferty).

Any animal species of immunoglobulin can be used in the invention.Non-limiting immunoglobulins useful in the present invention can be ofmurine, primate, or human origin. If the fusion protein is intended forhuman use, a chimeric form of immunoglobulin may be used wherein theconstant regions of the immunoglobulin are from a human. A humanized orfully human form of the immunoglobulin can also be prepared inaccordance with methods well known in the art (see e. g. U.S. Pat. No.5,565,332 to Winter). Humanization may be achieved by various methodsincluding, but not limited to (a) grafting the non-human (e.g., donorantibody) CDRs onto human (e.g. recipient antibody) framework andconstant regions with or without retention of critical frameworkresidues (e.g. those that are important for retaining good antigenbinding affinity or antibody functions), (b) grafting only the non-humanspecificity-determining regions (SDRs or a-CDRs; the residues criticalfor the antibody-antigen interaction) onto human framework and constantregions, or (c) transplanting the entire non-human variable domains, but“cloaking” them with a human-like section by replacement of surfaceresidues. Humanized antibodies and methods of making them are reviewed,e.g., in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), andare further described, e.g., in Riechmann et al., Nature 332, 323-329(1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989);U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones etal., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81,6851-6855 (1984); Morrison and 0i, Adv Immunol 44, 65-92 (1988);Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005)(describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28, 489-498(1991) (describing “resurfacing”); Dall'Acqua et al., Methods 36, 43-60(2005) (describing “FR shuffling”); and Osbourn et al., Methods 36,61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260 (2000)(describing the “guided selection” approach to FR shuffling). Particularimmunoglobulins according to the invention are human immunoglobulins.

Human antibodies and human variable regions can be produced usingvarious techniques known in the art. Human antibodies are describedgenerally in van Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74(2001) and Lonberg, Curr Opin Immuno! 20, 450-459 (2008). Human variableregions can form part of and be derived from human monoclonal antibodiesmade by the hybridoma method (see e.g. Monoclonal Antibody ProductionTechniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York,1987)). Human antibodies and human variable regions may also be preparedby administering an immunogen to a transgenic animal that has beenmodified to produce intact human antibodies or intact antibodies withhuman variable regions in response to antigenic challenge (see e.g.Lonberg, Nat Biotech 23, 1117-1125 (2005). Human antibodies and humanvariable regions may also be generated by isolating Fv clone variableregion sequences selected from human-derived phage display libraries(see e.g., Hoogenboom et al. in Methods in Molecular Biology 178, 1-37(O′Brien et al., ed., Human Press, Totowa, NJ, 2001); and McCafferty etal., Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)).Phage typically display antibody fragments, either as single-chain Fv(scFv) fragments or as Fab fragments.

In certain aspects, the moieties capable of specific binding to a targetcell antigen (e.g. Fab fragments) comprised in the antigen bindingmolecules of the present invention are engineered to have enhancedbinding affinity according to, for example, the methods disclosed in PCTpublication WO 2012/020006 (see Examples relating to affinitymaturation) or U.S. Pat. Appl. Publ. No. 2004/0132066. The ability ofthe antigen binding molecules of the invention to bind to a specificantigenic determinant can be measured either through an enzyme-linkedimmunosorbent assay (ELISA) or other techniques familiar to one of skillin the art, e.g. surface plasmon resonance technique (Liljeblad, et al.,Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley,Endocr Res 28, 217-229 (2002)). Competition assays may be used toidentify an antigen binding molecule that competes with a referenceantibody for binding to a particular antigen. In certain embodiments,such a competing antigen binding molecule binds to the same epitope(e.g. a linear or a conformational epitope) that is bound by thereference antigen binding molecule. Detailed exemplary methods formapping an epitope to which an antigen binding molecule binds areprovided in Morris (1996) “Epitope Mapping Protocols”, in Methods inMolecular Biology vol. 66 (Humana Press, Totowa, N.J.). In an exemplarycompetition assay, immobilized antigen is incubated in a solutioncomprising a first labeled antigen binding molecule that binds to theantigen and a second unlabeled antigen binding molecule that is beingtested for its ability to compete with the first antigen bindingmolecule for binding to the antigen. The second antigen binding moleculemay be present in a hybridoma supernatant. As a control, immobilizedantigen is incubated in a solution comprising the first labeled antigenbinding molecule but not the second unlabeled antigen binding molecule.After incubation under conditions permissive for binding of the firstantibody to the antigen, excess unbound antibody is removed, and theamount of label associated with immobilized antigen is measured. If theamount of label associated with immobilized antigen is substantiallyreduced in the test sample relative to the control sample, then thatindicates that the second antigen binding molecule is competing with thefirst antigen binding molecule for binding to the antigen. See Harlowand Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.).

TNF ligand trimer-containing antigen binding molecules of the inventionprepared as described herein may be purified by art-known techniquessuch as high performance liquid chromatography, ion exchangechromatography, gel electrophoresis, affinity chromatography, sizeexclusion chromatography, and the like. The actual conditions used topurify a particular protein will depend, in part, on factors such as netcharge, hydrophobicity, hydrophilicity etc., and will be apparent tothose having skill in the art. For affinity chromatography purificationan antibody, ligand, receptor or antigen can be used to which the TNFligand trimer-containing antigen binding molecule binds. For example,for affinity chromatography purification of fusion proteins of theinvention, a matrix with protein A or protein G may be used. SequentialProtein A or G affinity chromatography and size exclusion chromatographycan be used to isolate an antigen binding molecule essentially asdescribed in the Examples. The purity of the TNF ligandtrimer-containing antigen binding molecule or fragments thereof can bedetermined by any of a variety of well-known analytical methodsincluding gel electrophoresis, high pressure liquid chromatography, andthe like. For example, the TNF ligand trimer-containing antigen bindingmolecules expressed as described in the Examples were shown to be intactand properly assembled as demonstrated by reducing and non-reducingSDS-PAGE.

Assays

The antigen binding molecules provided herein may be identified,screened for, or characterized for their physical/chemical propertiesand/or biological activities by various assays known in the art.

1. Affinity Assays

The affinity of the TNF family ligand trimer-containing antigen bindingmolecule provided herein for the corresponding TNF receptor can bedetermined in accordance with the methods set forth in the Examples bysurface plasmon resonance (SPR), using standard instrumentation such asa BIAcore instrument (GE Healthcare), and receptors or target proteinssuch as may be obtained by recombinant expression. The affinity of theTNF family ligand trimer-containing antigen binding molecule for thetarget cell antigen can also be determined by surface plasmon resonance(SPR), using standard instrumentation such as a BIAcore instrument (GEHealthcare), and receptors or target proteins such as may be obtained byrecombinant expression. A specific illustrative and exemplary embodimentfor measuring binding affinity is described in Example 4. According toone aspect, K_(D) is measured by surface plasmon resonance using aBIACORE® T100 machine (GE Healthcare) at 25° C.

2. Binding Assays and Other Assays

Binding of the TNF family ligand trimer-containing antigen bindingmolecule provided herein to the corresponding receptor expressing cellsmay be evaluated using cell lines expressing the particular receptor ortarget antigen, for example by flow cytometry (FACS). In one aspect,fresh peripheral blood mononuclear cells (PBMCs) expressing the TNFreceptor are used in the binding assay. These cells are used directlyafter isolation (naive PMBCs) or after stimulation (activated PMBCs). Inanother aspect, activated mouse splenocytes (expressing the TNF receptormolecule) were used to demonstrate the binding of the TNF family ligandtrimer-containing antigen binding molecule of the invention to thecorresponding TNF receptor expressing cells.

In a further aspect, cancer cell lines expressing the target cellantigen, for example FAP, were used to demonstrate the binding of theantigen binding molecules to the target cell antigen.

In another aspect, competition assays may be used to identify an antigenbinding molecule that competes with a specific antibody or antigenbinding molecule for binding to the target or TNF receptor,respectively. In certain embodiments, such a competing antigen bindingmolecule binds to the same epitope (e.g., a linear or a conformationalepitope) that is bound by a specific anti-target antibody or a specificanti-TNF receptor antibody. Detailed exemplary methods for mapping anepitope to which an antibody binds are provided in Morris (1996)“Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66(Humana Press, Totowa, N.J.).

3. Activity Assays

In one aspect, assays are provided for identifying TNF family ligandtrimer-containing antigen binding molecules that bind to a specifictarget cell antigen and to a specific TNF receptor having biologicalactivity. Biological activity may include, e.g., agonistic signallingthrough the TNF receptor on cells expressing the target cell antigen.TNF family ligand trimer-containing antigen binding molecules identifiedby the assays as having such biological activity in vitro are alsoprovided.

In certain aspects, a TNF family ligand trimer-containing antigenbinding molecule of the invention is tested for such biologicalactivity. Assays for detecting the biological activity of the moleculesof the invention are those described in Example 6. Furthermore, assaysfor detecting cell lysis (e.g. by measurement of LDH release), inducedapoptosis kinetics (e.g. by measurement of Caspase 3/7 activity) orapoptosis (e.g. using the TUNEL assay) are well known in the art. Inaddition the biological activity of such complexes can be assessed byevaluating their effects on survival, proliferation and lymphokinesecretion of various lymphocyte subsets such as NK cells, NKT-cells orγδ T-cells or assessing their capacity to modulate phenotype andfunction of antigen presenting cells such as dendritic cells,monocytes/macrophages or B-cells.

Pharmaceutical Compositions, Formulations and Routes of Administation

In a further aspect, the invention provides pharmaceutical compositionscomprising any of the TNF family ligand trimer-containing antigenbinding molecules provided herein, e.g., for use in any of the belowtherapeutic methods. In one embodiment, a pharmaceutical compositioncomprises any of the TNF family ligand trimer-containing antigen bindingmolecules provided herein and at least one pharmaceutically acceptableexcipient. In another embodiment, a pharmaceutical composition comprisesany of the TNF family ligand trimer-containing antigen binding moleculesprovided herein and at least one additional therapeutic agent, e.g., asdescribed below.

Pharmaceutical compositions of the present invention comprise atherapeutically effective amount of one or more TNF family ligandtrimer-containing antigen binding molecules dissolved or dispersed in apharmaceutically acceptable excipient. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that are generally non-toxic to recipients at the dosagesand concentrations employed, i.e. do not produce an adverse, allergic orother untoward reaction when administered to an animal, such as, forexample, a human, as appropriate. The preparation of a pharmaceuticalcomposition that contains at least one TNF family ligandtrimer-containing antigen binding molecule and optionally an additionalactive ingredient will be known to those of skill in the art in light ofthe present disclosure, as exemplified by Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, incorporated herein byreference. In particular, the compositions are lyophilized formulationsor aqueous solutions. As used herein, “pharmaceutically acceptableexcipient” includes any and all solvents, buffers, dispersion media,coatings, surfactants, antioxidants, preservatives (e.g. antibacterialagents, antifungal agents), isotonic agents, salts, stabilizers andcombinations thereof, as would be known to one of ordinary skill in theart.

Parenteral compositions include those designed for administration byinjection, e.g. subcutaneous, intradermal, intralesional, intravenous,intraarterial intramuscular, intrathecal or intraperitoneal injection.For injection, the TNF family ligand trimer-containing antigen bindingmolecules of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'solution, Ringer's solution, or physiological saline buffer. Thesolution may contain formulatory agents such as suspending, stabilizingand/or dispersing agents. Alternatively, the fusion proteins may be inpowder form for constitution with a suitable vehicle, e.g., sterilepyrogen-free water, before use. Sterile injectable solutions areprepared by incorporating the fusion proteins of the invention in therequired amount in the appropriate solvent with various of the otheringredients enumerated below, as required. Sterility may be readilyaccomplished, e.g., by filtration through sterile filtration membranes.Generally, dispersions are prepared by incorporating the varioussterilized active ingredients into a sterile vehicle which contains thebasic dispersion medium and/or the other ingredients. In the case ofsterile powders for the preparation of sterile injectable solutions,suspensions or emulsion, the preferred methods of preparation arevacuum-drying or freeze-drying techniques which yield a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered liquid medium thereof. The liquid mediumshould be suitably buffered if necessary and the liquid diluent firstrendered isotonic prior to injection with sufficient saline or glucose.The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein. Suitable pharmaceuticallyacceptable excipients include, but are not limited to: buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Aqueous injectionsuspensions may contain compounds which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, dextran,or the like. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(18th Ed. Mack Printing Company, 1990). Sustained-release preparationsmay be prepared. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe polypeptide, which matrices are in the form of shaped articles, e.g.films, or microcapsules. In particular embodiments, prolonged absorptionof an injectable composition can be brought about by the use in thecompositions of agents delaying absorption, such as, for example,aluminum monostearate, gelatin or combinations thereof.

Exemplary pharmaceutically acceptable excipients herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

In addition to the compositions described previously, the fusionproteins may also be formulated as a depot preparation. Such long actingformulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the fusion proteins may be formulated with suitablepolymeric or hydrophobic materials (for example as emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions comprising the fusion proteins of theinvention may be manufactured by means of conventional mixing,dissolving, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Pharmaceutical compositions may be formulated in conventionalmanner using one or more physiologically acceptable carriers, diluents,excipients or auxiliaries which facilitate processing of the proteinsinto preparations that can be used pharmaceutically. Proper formulationis dependent upon the route of administration chosen.

The TNF family ligand trimer-containing antigen binding molecules may beformulated into a composition in a free acid or base, neutral or saltform. Pharmaceutically acceptable salts are salts that substantiallyretain the biological activity of the free acid or base. These includethe acid addition salts, e.g. those formed with the free amino groups ofa proteinaceous composition, or which are formed with inorganic acidssuch as for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric or mandelic acid. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas for example, sodium, potassium, ammonium, calcium or ferrichydroxides; or such organic bases as isopropylamine, trimethylamine,histidine or procaine. Pharmaceutical salts tend to be more soluble inaqueous and other protic solvents than are the corresponding free baseforms.

The composition herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such active ingredients are suitably present in combination inamounts that are effective for the purpose intended.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

Therapeutic Methods and Compositions

Any of the TNF family ligand trimer-containing antigen binding moleculesprovided herein may be used in therapeutic methods.

For use in therapeutic methods, TNF family ligand trimer-containingantigen binding molecules of the invention can be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners.

In one aspect, TNF family ligand trimer-containing antigen bindingmolecules of the invention for use as a medicament are provided. Infurther aspects, TNF family ligand trimer-containing antigen bindingmolecules of the invention for use in treating a disease, in particularfor use in the treatment of cancer, are provided. In certain aspects,TNF family ligand trimer-containing antigen binding molecules of theinvention for use in a method of treatment are provided. In one aspect,the invention provides a TNF family ligand trimer-containing antigenbinding molecule as described herein for use in the treatment of adisease in an individual in need thereof. In certain aspects, theinvention provides a TNF family ligand trimer-containing antigen bindingmolecule for use in a method of treating an individual having a diseasecomprising administering to the individual a therapeutically effectiveamount of the fusion protein. In certain aspects, the disease to betreated is cancer. Examples of cancers include solid tumors, bladdercancer, renal cell carcinoma, brain cancer, head and neck cancer,pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterinecancer, cervical cancer, endometrial cancer, esophageal cancer, coloncancer, colorectal cancer, rectal cancer, gastric cancer, prostatecancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer,and kidney cancer, melanoma, B-cell lymphoma, B-cell leukemia,non-Hodgkin lymphoma and acute lymphoblastic leukemia. Thus, a TNFfamily ligand trimer-containing antigen binding molecule as describedherein for use in the treatment of cancer is provided. The subject,patient, or “individual” in need of treatment is typically a mammal,more specifically a human.

In another aspect, provided is a TNF family ligand trimer-containingantigen binding molecule as described herein for use in the treatment ofinfectious diseases, in particular for the treatment of viralinfections. In a further aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as described herein for usein the treatment of autoimmune diseases such as for example Lupusdisease.

In one aspect, provided is a TNF family ligand trimer-containing antigenbinding molecule according to the invention for use in treating head andneck squamous cell carcinoma (HNSCC), breast cancer, colorectal cancer(CRC), pancreatic cancer (PAC), gastric cancer, non-small-cell lungcarcinoma (NSCLC) and Mesothelioma, wherein the target cell antigen isFAP.

In a further aspect, the invention relates to the use of a TNF familyligand trimer-containing antigen binding molecule in the manufacture orpreparation of a medicament for the treatment of a disease in anindividual in need thereof. In one aspect, the medicament is for use ina method of treating a disease comprising administering to an individualhaving the disease a therapeutically effective amount of the medicament.In certain embodiments the disease to be treated is a proliferativedisorder, particularly cancer. Thus, in one aspect, the inventionrelates to the use of a TNF family ligand trimer-containing antigenbinding molecule of the invention in the manufacture or preparation of amedicament for the treatment of cancer. Examples of cancers includesolid tumors, bladder cancer, renal cell carcinoma, brain cancer, headand neck cancer, pancreatic cancer, lung cancer, breast cancer, ovariancancer, uterine cancer, cervical cancer, endometrial cancer, esophagealcancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer,prostate cancer, blood cancer, skin cancer, squamous cell carcinoma,bone cancer, and kidney cancer, melanoma, B-cell lymphoma, B-cellleukemia, non-Hodgkin lymphoma and acute lymphoblastic leukemia. Othercell proliferation disorders that can be treated using a TNF familyligand trimer-containing antigen binding molecule of the presentinvention include, but are not limited to neoplasms located in the:abdomen, bone, breast, digestive system, liver, pancreas, peritoneum,endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary,thymus, thyroid), eye, head and neck, nervous system (central andperipheral), lymphatic system, pelvic, skin, soft tissue, spleen,thoracic region, and urogenital system. Also included are pre-cancerousconditions or lesions and cancer metastases. In certain embodiments thecancer is chosen from the group consisting of renal cell cancer, skincancer, lung cancer, colorectal cancer, breast cancer, brain cancer,head and neck cancer. A skilled artisan may recognize that in some casesthe TNF family ligand trimer-containing antigen binding molecule may notprovide a cure but may only provide partial benefit. In some aspects, aphysiological change having some benefit is also consideredtherapeutically beneficial. Thus, in some aspects, an amount of TNFfamily ligand trimer-containing antigen binding molecule that provides aphysiological change is considered an “effective amount” or a“therapeutically effective amount”.

In a further aspect, the invention relates to the use of a TNF familyligand trimer-containing antigen binding molecule as described herein inthe manufacture or preparation of a medicament for the treatment ofinfectious diseases, in particular for the treatment of viral infectionsor for the treatment of autoimmune diseases, for example Lupus disease.

In a further aspect, the invention provides a method for treating adisease in an individual, comprising administering to said individual atherapeutically effective amount of a TNF family ligandtrimer-containing antigen binding molecule of the invention. In oneaspect a composition is administered to said individual, comprising afusion protein of the invention in a pharmaceutically acceptable form.In certain aspects, the disease to be treated is a proliferativedisorder. In a particular aspect, the disease is cancer. In anotheraspect, the disease is an infectious disease or an autoimmune disease.In certain aspects, the method further comprises administering to theindividual a therapeutically effective amount of at least one additionaltherapeutic agent, e.g. an anti-cancer agent if the disease to betreated is cancer. An “individual” according to any of the aboveembodiments may be a mammal, preferably a human.

For the prevention or treatment of disease, the appropriate dosage of aTNF family ligand trimer-containing antigen binding molecule of theinvention (when used alone or in combination with one or more otheradditional therapeutic agents) will depend on the type of disease to betreated, the route of administration, the body weight of the patient,the type of fusion protein, the severity and course of the disease,whether the fusion protein is administered for preventive or therapeuticpurposes, previous or concurrent therapeutic interventions, thepatient's clinical history and response to the fusion protein, and thediscretion of the attending physician. The practitioner responsible foradministration will, in any event, determine the concentration of activeingredient(s) in a composition and appropriate dose(s) for theindividual subject. Various dosing schedules including but not limitedto single or multiple administrations over various time-points, bolusadministration, and pulse infusion are contemplated herein.

The TNF family ligand trimer-containing antigen binding molecule issuitably administered to the patient at one time or over a series oftreatments. Depending on the type and severity of the disease, about 1μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of TNF family ligandtrimer-containing antigen binding molecule can be an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. One typicaldaily dosage might range from about 1 μg/kg to 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentwould generally be sustained until a desired suppression of diseasesymptoms occurs. One exemplary dosage of the fusion protein would be inthe range from about 0.005 mg/kg to about 10 mg/kg. In other examples, adose may also comprise from about 1 μg/kg body weight, about 5 μg/kgbody weight, about 10 μg/kg body weight, about 50 μg/kg body weight,about 100 μg/kg body weight, about 200 μg/kg body weight, about 350μg/kg body weight, about 500 μg/kg body weight, about 1 mg/kg bodyweight, about 5 mg/kg body weight, about 10 mg/kg body weight, about 50mg/kg body weight, about 100 mg/kg body weight, about 200 mg/kg bodyweight, about 350 mg/kg body weight, about 500 mg/kg body weight, toabout 1000 mg/kg body weight or more per administration, and any rangederivable therein. In examples of a derivable range from the numberslisted herein, a range of about 5 mg/kg body weight to about 100 mg/kgbody weight, about 5 μg/kg body weight to about 500 mg/kg body weightetc., can be administered, based on the numbers described above. Thus,one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg(or any combination thereof) may be administered to the patient. Suchdoses may be administered intermittently, e.g. every week or every threeweeks (e.g. such that the patient receives from about two to abouttwenty, or e.g. about six doses of the fusion protein). An initialhigher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays.

The TNF family ligand trimer-containing antigen binding molecules of theinvention will generally be used in an amount effective to achieve theintended purpose. For use to treat or prevent a disease condition, theTNF family ligand trimer-containing antigen binding molecules of theinvention, or pharmaceutical compositions thereof, are administered orapplied in a therapeutically effective amount. Determination of atherapeutically effective amount is well within the capabilities ofthose skilled in the art, especially in light of the detailed disclosureprovided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays, such as cell culture assays. Adose can then be formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀ as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the TNF family ligand trimer-containing antigen bindingmolecules which are sufficient to maintain therapeutic effect. Usualpatient dosages for administration by injection range from about 0.1 to50 mg/kg/day, typically from about 0.5 to 1 mg/kg/day. Therapeuticallyeffective plasma levels may be achieved by administering multiple doseseach day. Levels in plasma may be measured, for example, by HPLC.

In cases of local administration or selective uptake, the effectivelocal concentration of the TNF family ligand trimer-containing antigenbinding molecule may not be related to plasma concentration. One skilledin the art will be able to optimize therapeutically effective localdosages without undue experimentation.

A therapeutically effective dose of the TNF family ligandtrimer-containing antigen binding molecules described herein willgenerally provide therapeutic benefit without causing substantialtoxicity. Toxicity and therapeutic efficacy of a fusion protein can bedetermined by standard pharmaceutical procedures in cell culture orexperimental animals. Cell culture assays and animal studies can be usedto determine the LD₅₀ (the dose lethal to 50% of a population) and theED₅₀ (the dose therapeutically effective in 50% of a population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, which can be expressed as the ratio LD₅₀/ED₅₀. TNF family ligandtrimer-containing antigen binding molecules that exhibit largetherapeutic indices are preferred. In one embodiment, the TNF familyligand trimer-containing antigen binding molecule according to thepresent invention exhibits a high therapeutic index. The data obtainedfrom cell culture assays and animal studies can be used in formulating arange of dosages suitable for use in humans. The dosage lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage may vary within this range dependingupon a variety of factors, e.g., the dosage form employed, the route ofadministration utilized, the condition of the subject, and the like. Theexact formulation, route of administration and dosage can be chosen bythe individual physician in view of the patient's condition (see, e.g.,Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch.1, p. 1, incorporated herein by reference in its entirety).

The attending physician for patients treated with fusion proteins of theinvention would know how and when to terminate, interrupt, or adjustadministration due to toxicity, organ dysfunction, and the like.Conversely, the attending physician would also know to adjust treatmentto higher levels if the clinical response were not adequate (precludingtoxicity). The magnitude of an administered dose in the management ofthe disorder of interest will vary with the severity of the condition tobe treated, with the route of administration, and the like. The severityof the condition may, for example, be evaluated, in part, by standardprognostic evaluation methods. Further, the dose and perhaps dosefrequency will also vary according to the age, body weight, and responseof the individual patient.

Other Agents and Treatments

The TNF family ligand trimer-containing antigen binding molecules of theinvention may be administered in combination with one or more otheragents in therapy. For instance, a fusion protein of the invention maybe co-administered with at least one additional therapeutic agent. Theterm “therapeutic agent” encompasses any agent that can be administeredfor treating a symptom or disease in an individual in need of suchtreatment. Such additional therapeutic agent may comprise any activeingredients suitable for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. In certain embodiments, an additional therapeuticagent is another anti-cancer agent.

Such other agents are suitably present in combination in amounts thatare effective for the purpose intended. The effective amount of suchother agents depends on the amount of fusion protein used, the type ofdisorder or treatment, and other factors discussed above. The TNF familyligand trimer-containing antigen binding molecules are generally used inthe same dosages and with administration routes as described herein, orabout from 1 to 99% of the dosages described herein, or in any dosageand by any route that is empirically/clinically determined to beappropriate.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate compositions), and separate administration, in which case,administration of the TNF family ligand trimer-containing antigenbinding molecule of the invention can occur prior to, simultaneously,and/or following, administration of the additional therapeutic agentand/or adjuvant.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper that ispierceable by a hypodermic injection needle). At least one active agentin the composition is a TNF ligand trimer-containing antigen bindingmolecule of the invention.

The label or package insert indicates that the composition is used fortreating the condition of choice. Moreover, the article of manufacturemay comprise (a) a first container with a composition contained therein,wherein the composition comprises a TNF ligand trimer-containing antigenbinding molecule of the invention; and (b) a second container with acomposition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition.

Alternatively, or additionally, the article of manufacture may furthercomprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

TABLE C (Sequences): SEQ ID NO: Name Sequence   1 Human (hu) 4-1BBL (71-REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP 254)LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVY YVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLP SPRSE   2 hu 4-1BBL (85-254)LDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSL TGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQ LTQGATVLGLFRVTPEIPAGLPSPRSE   3hu 4-1BBL (80-254) DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARAR HAWQLTQGATVLGLFRVTPEIPAGLPSPRSE   4hu 4-1BBL (52-254) PWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGL SYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQG ATVLGLFRVTPEIPAGLPSPRSE   5trimeric hu 4-1BBL (71- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP254) connected by (G4S)₂ LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVY linkerYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGF QGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDP GLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEAR ARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE  6 hu OX40L (51-183) QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSL DDFHVNGGELILIHQNPGEFCVL   7hu OX40L (52-183) VSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLD DFHVNGGELILIHQNPGEFCVL   8trimeric hu OX40L (51-183) QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKconnected by (G4S)₂ linker VQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSL DDFHVNGGELILIHQNPGEFCVLGGGGSGGGGSQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDF HVNGGELILIHQNPGEFCVLGGGGSGGGGSQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVN GGELILIHQNPGEFCVL   9FAP(28H1) CDR-H1 SHAMS  10 FAP(28H1) CDR-H2 AIWASGEQYYADSVKG  11FAP(28H1) CDR-H3 GWLGNFDY  12 FAP(28H1) CDR-L1 RASQSVSRSYLA  13FAP(28H1) CDR-L2 GASTRAT  14 FAP(28H1) CDR-L3 QQGQVIPPT  15trimeric hu 4-1BBL (71- See Table 1 254) Fc knob chain  16 FAP(28H1) VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHA MSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK GWLGNFDYWGQGTLVTVSS  17 FAP(28H1) VLEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLA WYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGT KVEIK  18 FAP(28H1) Fc hole chainSee Table 1  19 FAP(28H1) light chain See Table 1  20trimeric hu OX40L (51- See Table 27 183)-Fc knob chain  21Human (hu) FAP UniProt no. Q12884  22 hu FAP ectodomain + polyRPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWIS lys-tag + his₆-tagGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVL SICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAE YFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFS LSDGKKKKKKGHHHHHH  23nucleotide sequence CGCCCTTCAAGAGTTCATAACTCTGAAGAAAATAChu FAP ectodomain + poly- AATGAGAGCACTCACACTGAAGGATATTTTAAATGlys-tag+ his₆-tag GAACATTTTCTTATAAAACATTTTTTCCAAACTGGATTTCAGGACAAGAATATCTTCATCAATCTGCAGAT AACAATATAGTACTTTATAATATTGAAACAGGACAATCATATACCATTTTGAGTAATAGAACCATGAAAA GTGTGAATGCTTCAAATTACGGCTTATCACCTGATCGGCAATTTGTATATCTAGAAAGTGATTATTCAAA GCTTTGGAGATACTCTTACACAGCAACATATTACATCTATGACCTTAGCAATGGAGAATTTGTAAGAGGA AATGAGCTTCCTCGTCCAATTCAGTATTTATGCTGGTCGCCTGTTGGGAGTAAATTAGCATATGTCTATCA AAACAATATCTATTTGAAACAAAGACCAGGAGATCCACCTTTTCAAATAACATTTAATGGAAGAGAAAA TAAAATATTTAATGGAATCCCAGACTGGGTTTATGAAGAGGAAATGCTTGCTACAAAATATGCTCTCTGG TGGTCTCCTAATGGAAAATTTTTGGCATATGCGGAATTTAATGATACGGATATACCAGTTATTGCCTATTC CTATTATGGCGATGAACAATATCCTAGAACAATAAATATTCCATACCCAAAGGCTGGAGCTAAGAATCCC GTTGTTCGGATATTTATTATCGATACCACTTACCCTGCGTATGTAGGTCCCCAGGAAGTGCCTGTTCCAGC AATGATAGCCTCAAGTGATTATTATTTCAGTTGGCTCACGTGGGTTACTGATGAACGAGTATGTTTGCAG TGGCTAAAAAGAGTCCAGAATGTTTCGGTCCTGTCTATATGTGACTTCAGGGAAGACTGGCAGACATGGG ATTGTCCAAAGACCCAGGAGCATATAGAAGAAAGCAGAACTGGATGGGCTGGTGGATTCTTTGTTTCAA CACCAGTTTTCAGCTATGATGCCATTTCGTACTACAAAATATTTAGTGACAAGGATGGCTACAAACATATT CACTATATCAAAGACACTGTGGAAAATGCTATTCAAATTACAAGTGGCAAGTGGGAGGCCATAAATATA TTCAGAGTAACACAGGATTCACTGTTTTATTCTAGCAATGAATTTGAAGAATACCCTGGAAGAAGAAAC ATCTACAGAATTAGCATTGGAAGCTATCCTCCAAGCAAGAAGTGTGTTACTTGCCATCTAAGGAAAGAAA GGTGCCAATATTACACAGCAAGTTTCAGCGACTACGCCAAGTACTATGCACTTGTCTGCTACGGCCCAGG CATCCCCATTTCCACCCTTCATGATGGACGCACTGATCAAGAAATTAAAATCCTGGAAGAAAACAAGGA ATTGGAAAATGCTTTGAAAAATATCCAGCTGCCTAAAGAGGAAATTAAGAAACTTGAAGTAGATGAAAT TACTTTATGGTACAAGATGATTCTTCCTCCTCAATTTGACAGATCAAAGAAGTATCCCTTGCTAATTCAAG TGTATGGTGGTCCCTGCAGTCAGAGTGTAAGGTCTGTATTTGCTGTTAATTGGATATCTTATCTTGCAAGT AAGGAAGGGATGGTCATTGCCTTGGTGGATGGTCGAGGAACAGCTTTCCAAGGTGACAAACTCCTCTATG CAGTGTATCGAAAGCTGGGTGTTTATGAAGTTGAAGACCAGATTACAGCTGTCAGAAAATTCATAGAAAT GGGTTTCATTGATGAAAAAAGAATAGCCATATGGGGCTGGTCCTATGGAGGATACGTTTCATCACTGGCC CTTGCATCTGGAACTGGTCTTTTCAAATGTGGTATAGCAGTGGCTCCAGTCTCCAGCTGGGAATATTACGC GTCTGTCTACACAGAGAGATTCATGGGTCTCCCAACAAAGGATGATAATCTTGAGCACTATAAGAATTCA ACTGTGATGGCAAGAGCAGAATATTTCAGAAATGTAGACTATCTTCTCATCCACGGAACAGCAGATGATA ATGTGCACTTTCAAAACTCAGCACAGATTGCTAAAGCTCTGGTTAATGCACAAGTGGATTTCCAGGCAAT GTGGTACTCTGACCAGAACCACGGCTTATCCGGCCTGTCCACGAACCACTTATACACCCACATGACCCAC TTCCTAAAGCAGTGTTTCTCTTTGTCAGACGGCAAAAAGAAAAAGAAAAAGGGCCACCACCATCACCAT CAC  24 mouse FAP UniProt no. P97321 25 Murine FAP RPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWISectodomain + poly-lys- EQEYLHQSEDDNIVFYNIETRESYIILSNSTMKSVNATtag + his₆-tag DYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITYTGRENRIFNGIPDWVYEEEMLATKYALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVL SICDFREDWHAWECPKNQEHVEESRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAIYIFRVTQDSLFYSSNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIALVDGRGTAFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARA EYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGILSGRSQNHLYTHMTHFLKQCF SLSDGKKKKKKGHHHHHH  26nucleotide sequence CGTCCCTCAAGAGTTTACAAACCTGAAGGAAACAC Murine FAPAAAGAGAGCTCTTACCTTGAAGGATATTTTAAATG ectodomain + poly-lys-GAACATTCTCATATAAAACATATTTTCCCAACTGG tag + his₆-tagATTTCAGAACAAGAATATCTTCATCAATCTGAGGA TGATAACATAGTATTTTATAATATTGAAACAAGAGAATCATATATCATTTTGAGTAATAGCACCATGAAA AGTGTGAATGCTACAGATTATGGTTTGTCACCTGATCGGCAATTTGTGTATCTAGAAAGTGATTATTCAA AGCTCTGGCGATATTCATACACAGCGACATACTACATCTACGACCTTCAGAATGGGGAATTTGTAAGAGG ATACGAGCTCCCTCGTCCAATTCAGTATCTATGCTGGTCGCCTGTTGGGAGTAAATTAGCATATGTATAT CAAAACAATATTTATTTGAAACAAAGACCAGGAGATCCACCTTTTCAAATAACTTATACTGGAAGAGAA AATAGAATATTTAATGGAATACCAGACTGGGTTTATGAAGAGGAAATGCTTGCCACAAAATATGCTCTTT GGTGGTCTCCAGATGGAAAATTTTTGGCATATGTAGAATTTAATGATTCAGATATACCAATTATTGCCTA TTCTTATTATGGTGATGGACAGTATCCTAGAACTATAAATATTCCATATCCAAAGGCTGGGGCTAAGAAT CCGGTTGTTCGTGTTTTTATTGTTGACACCACCTACCCTCACCACGTGGGCCCAATGGAAGTGCCAGTTCC AGAAATGATAGCCTCAAGTGACTATTATTTCAGCTGGCTCACATGGGTGTCCAGTGAACGAGTATGCTTG CAGTGGCTAAAAAGAGTGCAGAATGTCTCAGTCCTGTCTATATGTGATTTCAGGGAAGACTGGCATGCAT GGGAATGTCCAAAGAACCAGGAGCATGTAGAAGAAAGCAGAACAGGATGGGCTGGTGGATTCTTTGTTT CGACACCAGCTTTTAGCCAGGATGCCACTTCTTACTACAAAATATTTAGCGACAAGGATGGTTACAAACA TATTCACTACATCAAAGACACTGTGGAAAATGCTATTCAAATTACAAGTGGCAAGTGGGAGGCCATATAT ATATTCCGCGTAACACAGGATTCACTGTTTTATTCTAGCAATGAATTTGAAGGTTACCCTGGAAGAAGAA ACATCTACAGAATTAGCATTGGAAACTCTCCTCCGAGCAAGAAGTGTGTTACTTGCCATCTAAGGAAAGA AAGGTGCCAATATTACACAGCAAGTTTCAGCTACAAAGCCAAGTACTATGCACTCGTCTGCTATGGCCCT GGCCTCCCCATTTCCACCCTCCATGATGGCCGCACAGACCAAGAAATACAAGTATTAGAAGAAAACAAA GAACTGGAAAATTCTCTGAGAAATATCCAGCTGCCTAAAGTGGAGATTAAGAAGCTCAAAGACGGGGGA CTGACTTTCTGGTACAAGATGATTCTGCCTCCTCAGTTTGACAGATCAAAGAAGTACCCTTTGCTAATTCA AGTGTATGGTGGTCCTTGTAGCCAGAGTGTTAAGTCTGTGTTTGCTGTTAATTGGATAACTTATCTCGCAA GTAAGGAGGGGATAGTCATTGCCCTGGTAGATGGTCGGGGCACTGCTTTCCAAGGTGACAAATTCCTGCA TGCCGTGTATCGAAAACTGGGTGTATATGAAGTTGAGGACCAGCTCACAGCTGTCAGAAAATTCATAGA AATGGGTTTCATTGATGAAGAAAGAATAGCCATATGGGGCTGGTCCTACGGAGGTTATGTTTCATCCCTG GCCCTTGCATCTGGAACTGGTCTTTTCAAATGTGGCATAGCAGTGGCTCCAGTCTCCAGCTGGGAATATT ACGCATCTATCTACTCAGAGAGATTCATGGGCCTCCCAACAAAGGACGACAATCTCGAACACTATAAAA ATTCAACTGTGATGGCAAGAGCAGAATATTTCAGAAATGTAGACTATCTTCTCATCCACGGAACAGCAGA TGATAATGTGCACTTTCAGAACTCAGCACAGATTGCTAAAGCTTTGGTTAATGCACAAGTGGATTTCCAG GCGATGTGGTACTCTGACCAGAACCATGGTATATTATCTGGGCGCTCCCAGAATCATTTATATACCCACA TGACGCACTTCCTCAAGCAATGCTTTTCTTTATCAGACGGCAAAAAGAAAAAGAAAAAGGGCCACCACCA TCACCATCAC  27 Cynomolgus FAPRPPRVHNSEENTMRALTLKDILNGTFSYKTFFPNWIS ectodomain + poly-lys-GQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNA tag + his₆-tagSNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPFVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVL SICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEDYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAP VSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNA QVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSDGKKKKKKGHHHHHH  28 nucleotide sequenceCGCCCTCCAAGAGTTCATAACTCTGAAGAAAATAC Cynomolgus FAPAATGAGAGCACTCACACTGAAGGATATTTTAAATG ectodomain + poly-lys-GGACATTTTCTTATAAAACATTTTTTCCAAACTGGA tag + his₆-tagTTTCAGGACAAGAATATCTTCATCAATCTGCAGAT AACAATATAGTACTTTATAATATTGAAACAGGACAATCATATACCATTTTGAGTAACAGAACCATGAAAA GTGTGAATGCTTCAAATTATGGCTTATCACCTGATCGGCAATTTGTATATCTAGAAAGTGATTATTCAAA GCTTTGGAGATACTCTTACACAGCAACATATTACATCTATGACCTTAGCAATGGAGAATTTGTAAGAGGA AATGAGCTTCCTCGTCCAATTCAGTATTTATGCTGGTCGCCTGTTGGGAGTAAATTAGCATATGTCTATCA AAACAATATCTATTTGAAACAAAGACCAGGAGATCCACCTTTTCAAATAACATTTAATGGAAGAGAAAA TAAAATATTTAATGGAATCCCAGACTGGGTTTATGAAGAGGAAATGCTTGCTACAAAATATGCTCTCTGG TGGTCTCCTAATGGAAAATTTTTGGCATATGCGGAATTTAATGATACAGATATACCAGTTATTGCCTATTC CTATTATGGCGATGAACAATATCCCAGAACAATAAATATTCCATACCCAAAGGCCGGAGCTAAGAATCCT TTTGTTCGGATATTTATTATCGATACCACTTACCCTGCGTATGTAGGTCCCCAGGAAGTGCCTGTTCCAGC AATGATAGCCTCAAGTGATTATTATTTCAGTTGGCTCACGTGGGTTACTGATGAACGAGTATGTTTGCAG TGGCTAAAAAGAGTCCAGAATGTTTCGGTCTTGTCTATATGTGATTTCAGGGAAGACTGGCAGACATGGG ATTGTCCAAAGACCCAGGAGCATATAGAAGAAAGCAGAACTGGATGGGCTGGTGGATTCTTTGTTTCAA CACCAGTTTTCAGCTATGATGCCATTTCATACTACAAAATATTTAGTGACAAGGATGGCTACAAACATATT CACTATATCAAAGACACTGTGGAAAATGCTATTCAAATTACAAGTGGCAAGTGGGAGGCCATAAATATA TTCAGAGTAACACAGGATTCACTGTTTTATTCTAGCAATGAATTTGAAGATTACCCTGGAAGAAGAAAC ATCTACAGAATTAGCATTGGAAGCTATCCTCCAAGCAAGAAGTGTGTTACTTGCCATCTAAGGAAAGAAA GGTGCCAATATTACACAGCAAGTTTCAGCGACTACGCCAAGTACTATGCACTTGTCTGCTATGGCCCAGG CATCCCCATTTCCACCCTTCATGACGGACGCACTGATCAAGAAATTAAAATCCTGGAAGAAAACAAGGA ATTGGAAAATGCTTTGAAAAATATCCAGCTGCCTAAAGAGGAAATTAAGAAACTTGAAGTAGATGAAAT TACTTTATGGTACAAGATGATTCTTCCTCCTCAATTTGACAGATCAAAGAAGTATCCCTTGCTAATTCAAG TGTATGGTGGTCCCTGCAGTCAGAGTGTAAGGTCTGTATTTGCTGTTAATTGGATATCTTATCTTGCAAGT AAGGAAGGGATGGTCATTGCCTTGGTGGATGGTCGGGGAACAGCTTTCCAAGGTGACAAACTCCTGTATG CAGTGTATCGAAAGCTGGGTGTTTATGAAGTTGAAGACCAGATTACAGCTGTCAGAAAATTCATAGAAAT GGGTTTCATTGATGAAAAAAGAATAGCCATATGGGGCTGGTCCTATGGAGGATATGTTTCATCACTGGCC CTTGCATCTGGAACTGGTCTTTTCAAATGTGGGATAGCAGTGGCTCCAGTCTCCAGCTGGGAATATTACG CGTCTGTCTACACAGAGAGATTCATGGGTCTCCCAACAAAGGATGATAATCTTGAGCACTATAAGAATTC AACTGTGATGGCAAGAGCAGAATATTTCAGAAATGTAGACTATCTTCTCATCCACGGAACAGCAGATGA TAATGTGCACTTTCAAAACTCAGCACAGATTGCTAAAGCTCTGGTTAATGCACAAGTGGATTTCCAGGCA ATGTGGTACTCTGACCAGAACCACGGCTTATCCGGCCTGTCCACGAACCACTTATACACCCACATGACCC ACTTCCTAAAGCAGTGTTTCTCTTTGTCAGACGGCAAAAAGAAAAAGAAAAAGGGCCACCACCATCACC ATCAC  29 human CEAUniProt no. P06731  30 human MCSP UniProt no. Q6UVK1  31 human EGFRUniProt no. P00533  32 human CD19 UniProt no. P15391  33 human CD20Uniprot no. P11836  34 human CD33 UniProt no. P20138  35human Lymphotoxin α UniProt no. P01374  36 human TNF UniProt no. P01375 37 human Lymphotoxin β UniProt no. Q06643  38 human OX40LUniProt no. P23510  39 human CD40L UniProt no. P29965  40 human FasLUniProt no. P48023  41 human CD27L UniProt no. P32970  42 human CD30LUniProt no. P32971  43 human 4-1BBL UniProt no. P41273  44 human TRAILUniProt no. P50591  45 human RANKL UniProt no. O14788  46 human TWEAKUniProt no. O43508  47 human APRIL UniProt no. O75888  48 human BAFFUniProt no. Q9Y275  49 human LIGHT UniProt no. O43557  50 human TL1AUniProt no. O95150  51 human GITRL UniProt no. Q9UNG2  52human ectodysplasin A UniProt no. Q92838  53 hu 4-1BBL (50-254)ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLT GGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQL TQGATVLGLFRVTPEIPAGLPSPRSE  54Peptide linker (G4S)₂ GGGGSGGGGS  55 Peptide linker (SG4)₂ SGGGGSGGGG 56 Peptide linker G4(SG4)₂ GGGGSGGGGSGGGG  57 Peptide linker GSPGSSSSGS 58 Peptide linker (G4S)₄ GGGGSGGGGSGGGGSGGGGS  59 Peptide linkerGSGSGNGS  60 Peptide linker GGSGSGSG  61 Peptide linker GGSGSG  62Peptide linker GGSG  63 Peptide linker GGSGNGSG  64 Peptide linkerGGNGSGSG  65 Peptide linker GGNGSG  66 nucleotide sequence trimericSee Table 1 hu 4-1BBL (71-254) Fc knob chain  67 nucleotide sequenceSee Table 1 FAP(28H1) Fc hole chain  68 nucleotide sequence See Table 1FAP(28H1) light chain  69 nucleotide sequence DP47 See Table 19Fc hole chain  70 Nucleotide sequence See Table 19 DP47 light chain  71DP47 Fc hole chain See Table 19  72 DP47 light chain See Table 19  73Human 4-1BB ECD Uniprot No. Q07011, aa 24-186  74 Cynomolgus 4-1BB ECDaa 24-186  75 Murine 4-1BB ECD Uniprot No. P20334, aa 24-187  76nucleotide sequence Fc hole See Table 24 chain  77nucleotide sequence human See Table 24 4-1BB Fc knob chain  78nucleotide sequence See Table 24 cynomolgus 4-1BB Fc knob chain  79nucleotide sequence murine See Table 24 4-1BB Fc knob chain  80Fc hole chain See Table 24  81 human 4-1BB Fc knob chain See Table 24 82 cynomolgus 4-1BB Fc knob See Table 24 chain  83 murine 4-1BB Fc knobSee Table 24 chain  84 Avi tag GLNDI FEAQK IEWHE  85nucleotide sequence Human See Table 25 4-1BB His  86 Human 4-1BB HisSee Table 25  87 nucleotide sequence trimeric See Table 27hu OX40L (51-183) Fc knob chain  88 Human OX40 ECDUniprot No. P43489, aa 29-214  89 Cynomolgus OX40 ECD aa 29-214  90Murine OX40 ECD Uniprot No. P47741, aa 10-211  91nucleotide sequence human See Table 30 OX40 Fc knob chain  92nucleotide sequence See Table 30 cynomolgus OX40 Fc knob chain  93nucleotide sequence See Table 30 murine OX40 Fc knob chain  94human OX40 Fc knob chain See Table 30  95 cynomolgus OX40 Fe knobSee Table 30 chain  96 murine OX40 Fc knob chain See Table 30  97nucleotide sequence See Table 31 human OX40 His  98 human OX40 HisSee Table 31  99 Human (hu) 4-1BBL (71-REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP 248)LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVY YVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLG VHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGL100 hu 4-1BBL (85-248) LDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQ LTQGATVLGLFRVTPEIPAGL 101hu 4-1BBL (80-248) DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARAR HAWQLTQGATVLGLFRVTPEIPAGL 102hu 4-1BBL (52-248) PWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGL SYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQG ATVLGLFRVTPEIPAGL 103trimeric hu 4-1BBL (71- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP248) connected by (G4S)₂ LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVY linkerYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKE LVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRL LHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTG GLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLT QGATVLGLFRVTPEIPAGL 104FAP(4B9) CDR-H1 SYAMS 105 FAP(4B9) CDR-H2 AIIGSGASTYYADSVKG 106FAP(4B9) CDR-H3 GWFGGFNY 107 FAP(4B9) CDR-L1 RASQSVTSSYLA 108FAP(4B9) CDR-L2 VGSRRAT 109 FAP(4B9) CDR-L3 QQGIMLPPT 110 FAP(4B9) VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRD NSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS 111 FAP(4B9) VL EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIK112 CEA(sm9b) CDR-H1 DSYMH 113 CEA(sm9b) CDR-H2 WIDPENGDTEYAPKFQG 114CEA(sm9b) CDR-H3 GTPTGPYYFDY 115 CEA(sm9b) CDR-L1 SASS SVPYMH 116CEA(sm9b) CDR-L2 STSNLAS 117 CEA(sm9b) CDR-L3 QQRSSYPLT 118 CEA(sm9b) VHQVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMH WLRQGPGQCLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYYCNEGTPTGPYYF DYWGQGTLVTVSS 119 CEA(sm9b) VLENVLTQSPSSMSVSVGDRVTIACSASSSVPYMHWLQQKPGKSPKLLIYSTSNLASGVPSRFSGSGSGTDYSLTI SSVQPEDAATYYCQQRSSYPLTFGCGTKLEIK120 CD19 (8B8-018) CDR-H1 DYIMH 121 CD19 (8B8-018) CDR-H2YINPYNDGSKYTEKFQG 122 CD19 (8B8-018) CDR-H3 GTYYYGSALFDY 123CD19 (8B8-018) CDR-L1 KSSQSLENPNGNTYLN 124 CD19 (8B8-018) CDR-L2 RVSKRFS125 CD19 (8B8-018) CDR-L3 LQLTHVPYT 126 CD19 (8B8-018) VHQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMH WVRQAPGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGS ALFDYWGQGTTVTVSS 127CD19 (8B8-018) VL DIVMTQTPLSLSVTPGQPASISCKSSQSLENPNGNTYLNWYLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQLTHVPYTFGQGTKLEIK 128 CD19 (8B8-2B11) CDR-H1 DYIMH129 CD19 (8B8-2B11) CDR-H2 YINPYNDGSKYTEKFQG 130 CD19 (8B8-2B11) CDR-H3GTYYYGPQLFDY 131 CD19 (8B8-2B11) CDR-L1 KSSQSLETSTGTTYLN 132CD19 (8B8-2B11) CDR-L2 RVSKRFS 133 CD19 (8B8-2B11) CDR-L3 LQLLEDPYT 134CD19 (8B8-2B11) VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQAPGQGLEWMGYINPYNDGSKYTEKFQGRVT MTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGPQLFDYWGQGTTVTVSS 135 CD19 (8B8-2B11) VLDIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGTTYLNWYLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQLLEDPYTFGQGTKLEIK 136 G4S GGGGS 137nucleotide sequence of anti- Table 3 FAP(4B9) Fc hole chain 138nucleotide sequence of anti- Table 3 FAP(4B9) Fc knob chainfused to trimeric hu 4-1BBL (71-254) 139 nucleotide sequence of anti-Table 3 FAP(4B9) light chain 140 anti-FAP(4B9) Fc hole chain Table 3 141anti-FAP(4B9) Fc knob Table 3 chain fused to trimeric hu 4-1BBL (71-254) 142 anti-FAP(4B9) light chain Table 3 143nucleotide sequence of anti- Table 5 CEA(sm9b) Fc hole chain 144nucleotide sequence of anti- Table 5 CEA(sm9b) Fc knob chainfused to trimeric hu 4-1BBL (71-254) 145 nucleotide sequence of anti-Table 5 CEA(sm9b) light chain 146 anti-CEA(sm9b) Fc hole Table 5 chain147 anti-CEA(sm9b) Fc knob Table 5 chain fused to trimeric hu 4-1BBL (71-254) 148 anti-CEA(sm9b) light chain Table 5 149nucleotide sequence of anti- Table 7 CD19(8B8-018)Fc hole chain 150nucleotide sequence of anti- Table 7 CD19(8B8-018) Fc knobchain fused to trimeric hu 4- 1BBL (71-254) 151nucleotide sequence of anti- Table 7 CD19(8B8-018) light chain 152anti-CD19(8B8-018) Fc Table 7 hole chain 153 anti-CD19(8B8-018) FcTable 7 knob chain fused to trimeric hu 4-1BBL (71-254) 154anti-CD19(8B8-018) light Table 7 chain 155 nucleotide sequence of anti-Table 8 CD19(8B8-2B11) Fc hole chain 156 nucleotide sequence of anti-Table 8 CD19(8B8-2B11) Fc knob chain fused to trimeric hu 4-1BBL (71-254) 157 nucleotide sequence of anti- Table 8CD19(8B8-2B11) light chain 158 anti-CD19(8B8-2B11) Fc Table 8 hole chain159 anti-CD19(8B8-2B11) Fc Table 8 knob chain fused to trimerichu 4-1BBL (71-254) 160 anti-CD19(8B8-2B11) light Table 8 chain 161HVR-L1 anti-CD19(8B8) NSNGNT 162 HVR-H2 anti-CD19(8B8) KFNG 163Nucleotide sequence of Fc Table 13 hole chain with HYRF mutation 164Nucleotide sequence of Table 13 human CD19 antigen Fc knob chain avi tag165 Fc hole chain with HYRF Table 13 mutation 166 human CD19 antigen FcTable 13 knob chain avi tag 167 Nucleotide sequence of Table 13cynomolgus CD19 antigen Fc knob chain avi tag 168cynomolgus CD19 antigen Table 13 Fc knob chain avi tag 169Nucleotide sequence CD 19 GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGT (8B8) VHAAAGCCTGGGGCTTCAGTGAAGATGGCCTGCAAG Parental cloneGCTTCTGGATACACATTCACTGACTATATTATGCA CTGGGTGAAGCAGAAGACTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATGGTTCT AAGTACACTGAGAAGTTCAACGGCAAGGCCACACTGACTTCAGACAAATCTTCCATCACAGCCTACATG GAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGGGACCTATTATTATGGTA GCGCCCTCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCG 170 Nucleotide sequence CD 19GATGCTGTGATGACCCAAACTCCACTCTCCCTGCC (8B8) VLTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCA Parental cloneGGTCTAGTCAGAGCCTTGAAAACAGTAATGGAAA CACCTATTTGAACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAACTCCTGATCTACAGGGTTTCCAAA CGATTTTCTGGGGTCCTAGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCA GAGTGGAGGCTGAGGATTTGGGAGTTTATTTCTGCCTACAACTTACACATGTCCCGTACACGTTCGGAGG GGGGACCAAGCTGGAAATAAAA 171CD19 L1 reverse random see Table 15 172 CD19 L2 forward randomsee Table 15 173 CD19 H1 reverse random see Table 15 174CD19 H2 forward random see Table 15 175 CD19 H3 reverse constantsee Table 15 176 LMB3 see Table 15 177 CD19 L1 forward constantsee Table 16 178 CD19 L3 reverse random see Table 16 179CD19 L3 forward constant see Table 16 180 CD19 H3 reverse randomsee Table 16 181 nucleotide sequence of see Table 21DP47 Fc knob fused to trimeric hu 4-1BBL (71- 254) 182DP47 Fc knob fused to see Table 21 trimeric hu 4-1BBL (71- 254) 183CMV-derived peptide NLVPMVATV

In the following specific embodiments of the invention are listed:

-   -   A TNF family ligand trimer-containing antigen binding molecule        comprising        -   (a) at least one moiety capable of specific binding to a            target cell antigen,        -   (b) a polypeptide comprising three ectodomains of a TNF            ligand family member or fragments thereof that are connected            to each other, optionally by peptide linkers, and        -   (c) a Fc domain composed of a first and a second subunit            capable of stable association.    -   The TNF family ligand trimer-containing antigen binding molecule        comprising        -   (a) at least one moiety capable of specific binding to a            target cell antigen,        -   (b) a polypeptide comprising three ectodomains of a TNF            ligand family member or fragments thereof that are connected            to each other by peptide linkers and        -   (c) a Fc domain composed of a first and a second subunit            capable of stable association.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, comprising        -   (a) at least one moiety capable of specific binding to a            target cell antigen,        -   (b) a polypeptide comprising three ectodomains of a TNF            ligand family member or fragments thereof that are connected            to each other by peptide linkers and        -   (c) a Fc domain composed of a first and a second subunit            capable of stable association, wherein the polypeptide            comprising the three ectodomains of a TNF ligand family            member or fragments thereof that are connected to each other            by peptide linkers is fused to the N- or C-terminal amino            acid of one of the two subunits of the Fc domain, optionally            through a peptide linker.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the TNF ligand family member        costimulates human T-cell activation.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the TNF ligand family member is        selected from 4-1BBL and OX40L.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the TNF ligand family member is        4-1BBL.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the ectodomain of a TNF ligand        family member comprises the amino acid sequence selected from        the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3,        SEQ ID NO:4, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO:101 and SEQ        ID NO:102, particularly the amino acid sequence of SEQ ID NO:1        or SEQ ID NO:99.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the ectodomain of a TNF ligand        family member comprises the amino acid sequence selected from        the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3        and SEQ ID NO:4, particularly the amino acid sequence of SEQ ID        NO:1.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, comprising        -   (a) at least one moiety capable of specific binding to a            target cell antigen and        -   (b) a polypeptide comprising the amino acid sequence of SEQ            ID NO:5 or SEQ ID NO:103, and        -   (c) a Fc domain composed of a first and a second subunit            capable of stable association.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, comprising        -   (a) at least one moiety capable of specific binding to a            target cell antigen and        -   (b) a polypeptide comprising the amino acid sequence of SEQ            ID NO:5 and        -   (c) a Fc domain composed of a first and a second subunit            capable of stable association.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the TNF ligand family member is        OX40L.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the ectodomain of a TNF ligand        family member comprises the amino acid sequence selected from        the group consisting of SEQ ID NO:6 and SEQ ID NO:7,        particularly the amino acid sequence of SEQ ID NO:6.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, comprising        -   (a) at least one moiety capable of specific binding to a            target cell antigen and        -   (b) a polypeptide comprising the amino acid sequence of SEQ            ID NO:8, and        -   (c) a Fc domain composed of a first and a second subunit            capable of stable association.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the polypeptide comprising three        ectodomains of a TNF ligand family member or fragments thereof        is fused at the C-terminal amino acid to the N-terminal amino        acid of one of the subunits of the Fc domain.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the polypeptide comprising three        ectodomains of a TNF ligand family member or fragments thereof        is fused at the N-terminal amino acid to a C-terminal amino acid        of one of the subunits of the Fc domain.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the moiety capable of specific        binding to a target cell antigen is selected from the group        consisting of an antibody fragment, a Fab molecule, a crossover        Fab molecule, a single chain Fab molecule, a Fv molecule, a scFv        molecule, a single domain antibody, an aVH and a scaffold        antigen binding protein.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the moiety capable of specific        binding to a target cell antigen is a Fab molecule capable of        specific binding to a target cell antigen.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the target cell antigen is selected        from the group consisting of Fibroblast Activation Protein        (FAP), Melanoma-associated Chondroitin Sulfate Proteoglycan        (MCSP), Epidermal Growth Factor Receptor (EGFR),        Carcinoembryonic Antigen (CEA), CD19, CD20 and CD33.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the target cell antigen is        Fibroblast Activation Protein (FAP).    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the moiety capable of specific        binding to FAP comprises a VH domain comprising (i) CDR-H1        comprising the amino acid sequence of SEQ ID NO:9, (ii) CDR-H2        comprising the amino acid sequence of SEQ ID NO:10 and (iii)        CDR-H3 comprising the amino acid sequence of SEQ ID NO:11, and a        VL domain comprising (iv) CDR-L1 comprising the amino acid        sequence of SEQ ID NO:12, (v) CDR-L2 comprising the amino acid        sequence of SEQ ID NO:13 and (vi) CDR-L3 comprising the amino        acid sequence of SEQ ID NO:14.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the moiety capable of specific        binding to FAP comprises a VH domain domain comprising (i)        CDR-H1 comprising the amino acid sequence of SEQ ID NO:104, (ii)        CDR-H2 comprising the amino acid sequence of SEQ ID NO:105        and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID        NO:106, and a VL domain comprising (iv) CDR-L1 comprising the        amino acid sequence of SEQ ID NO:107, (v) CDR-L2 comprising the        amino acid sequence of SEQ ID NO:108 and (vi) CDR-L3 comprising        the amino acid sequence of SEQ ID NO:109.    -   The TNF family ligand trimer-containing antigen binding molecule        as described herein before, wherein the moiety capable of        specific binding to FAP comprises a variable heavy chain        comprising an amino acid sequence of SEQ ID NO:16 and a variable        light chain comprising an amino acid sequence of SEQ ID NO:17 or        wherein the moiety capable of specific binding to FAP comprises        a variable heavy chain comprising an amino acid sequence of SEQ        ID NO:110 and a variable light chain comprising an amino acid        sequence of SEQ ID NO:111.    -   The TNF family ligand trimer-containing antigen binding molecule        of any one of claims 1 to 15, wherein the target cell antigen is        CEA.    -   The TNF family ligand trimer-containing antigen binding molecule        as described herein before, wherein the moiety capable of        specific binding to CEA comprises a VH domain comprising (i)        CDR-H1 comprising the amino acid sequence of SEQ ID NO:112, (ii)        CDR-H2 comprising the amino acid sequence of SEQ ID NO:113        and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID        NO:114, and a VL domain comprising (iv) CDR-L1 comprising the        amino acid sequence of SEQ ID NO:115, (v) CDR-L2 comprising the        amino acid sequence of SEQ ID NO:116 and (vi) CDR-L3 comprising        the amino acid sequence of SEQ ID NO:117.    -   The TNF family ligand trimer-containing antigen binding molecule        as described herein before, wherein the moiety capable of        specific binding to CEA comprises a variable heavy chain        comprising an amino acid sequence of SEQ ID NO:118 and a        variable light chain comprising an amino acid sequence of SEQ ID        NO:119.    -   The TNF family ligand trimer-containing antigen binding molecule        as described herein before, wherein the target cell antigen is        CD19.    -   The TNF family ligand trimer-containing antigen binding molecule        as described herein before, wherein the moiety capable of        specific binding to CD19 comprises        -   (a) a VH domain comprising (i) CDR-H1 comprising the amino            acid sequence of SEQ ID NO:120, (ii) CDR-H2 comprising the            amino acid sequence of SEQ ID NO:121 and (iii) CDR-H3            comprising the amino acid sequence of SEQ ID NO:122, and a            VL domain comprising (iv) CDR-L1 comprising the amino acid            sequence of SEQ ID NO:123, (v) CDR-L2 comprising the amino            acid sequence of SEQ ID NO:124 and (vi) CDR-L3 comprising            the amino acid sequence of SEQ ID NO:125, or        -   (b) a VH domain comprising (i) CDR-H1 comprising the amino            acid sequence of SEQ ID NO:128, (ii) CDR-H2 comprising the            amino acid sequence of SEQ ID NO:129 and (iii) CDR-H3            comprising the amino acid sequence of SEQ ID NO:130, and a            VL domain comprising (iv) CDR-L1 comprising the amino acid            sequence of SEQ ID NO:131, (v) CDR-L2 comprising the amino            acid sequence of SEQ ID NO:132 and (vi) CDR-L3 comprising            the amino acid sequence of SEQ ID NO:133.    -   The TNF family ligand trimer-containing antigen binding molecule        as described herein before, wherein the moiety capable of        specific binding to CD19 comprises a variable heavy chain        comprising an amino acid sequence of SEQ ID NO:126 and a        variable light chain comprising an amino acid sequence of SEQ ID        NO:127 or wherein the moiety capable of specific binding to FAP        comprises a variable heavy chain comprising an amino acid        sequence of SEQ ID NO:134 and a variable light chain comprising        an amino acid sequence of SEQ ID NO:135.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the Fc domain is an IgG,        particularly an IgG1 Fc domain or an IgG4 Fc domain.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the Fc domain is an IgG1 Fc domain        and comprises amino acid substitutions at positions 234 and 235        (EU numbering) and/or 329 (EU numbering) of the IgG heavy        chains.    -   The TNF family ligand trimer-containing antigen binding molecule        as described herein before, comprising one moiety capable of        specific binding to a target cell antigen.    -   The TNF family ligand trimer-containing antigen binding molecule        as described herein before, wherein the antigen binding molecule        comprises        -   (i) a first heavy chain comprising the VH domain of a Fab            molecule capable of specific binding to a target cell            antigen,        -   (ii) a light chain comprising the VL domain of a Fab            molecule capable of specific binding to a target cell            antigen, and        -   (iii) a second heavy chain comprising the amino acid            sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8.    -   The TNF family ligand trimer-containing antigen binding molecule        as described herein before, wherein the antigen binding molecule        comprises        -   (i) a first heavy chain comprising a VH domain comprising an            amino acid sequence of SEQ ID NO:16 or a VH domain            comprising an amino acid sequence of SEQ ID NO:110,        -   (ii) a light chain comprising a VL domain comprising an            amino acid sequence of SEQ ID NO:17 or a VL domain            comprising an amino acid sequence of SEQ ID NO:111, and        -   (iii) a second heavy chain comprising the amino acid            sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8.    -   The TNF family ligand trimer-containing antigen binding molecule        as described herein before, wherein the antigen binding molecule        comprises        -   (i) a first heavy chain comprising a VH domain comprising an            amino acid sequence of SEQ ID NO:118,        -   (ii) a light chain comprising a VL domain comprising an            amino acid sequence of SEQ ID NO:119, and        -   (iii) a second heavy chain comprising the amino acid            sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8.    -   The TNF family ligand trimer-containing antigen binding molecule        as described herein before, wherein the antigen binding molecule        comprises        -   (i) a first heavy chain comprising a VH domain comprising an            amino acid sequence of SEQ ID NO:126 or a VH domain            comprising an amino acid sequence of SEQ ID NO:134,        -   (ii) a light chain comprising a VL domain comprising an            amino acid sequence of SEQ ID NO:127 or a VL domain            comprising an amino acid sequence of SEQ ID NO:135, and        -   (iii) a second heavy chain comprising the amino acid            sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8.    -   The TNF family ligand trimer-containing antigen binding molecule        as described herein before, comprising two moieties capable of        specific binding to a target cell antigen.    -   The TNF family ligand trimer-containing antigen binding molecule        as described herein before, wherein the antigen binding molecule        comprises        -   (i) a first heavy chain comprising a VH domain of a Fab            molecule capable of specific binding to a target cell            antigen,        -   (ii) two light chains comprising each the VL domain of a Fab            molecule capable of specific binding to a target cell            antigen, and        -   (iii) a second heavy chain comprising a VH domain of a Fab            molecule capable of specific binding to a target cell            antigen and the amino acid sequence of SEQ ID NO:5 or SEQ ID            NO:103 or SEQ ID NO:8.    -   The TNF family ligand trimer-containing antigen binding molecule        as described herein before, wherein the antigen binding molecule        comprises        -   (i) a first heavy chain comprising a VH domain comprising an            amino acid sequence of SEQ ID NO:16 or a VH domain            comprising an amino acid sequence of SEQ ID NO:110,        -   (ii) two light chains comprising each a VL domain comprising            an amino acid sequence of SEQ ID NO:17 or a VL domain            comprising an amino acid sequence of SEQ ID NO:111, and        -   (iii) a second heavy chain comprising a VH domain comprising            an amino acid sequence of SEQ ID NO:16 or a VH domain            comprising an amino acid sequence of SEQ ID NO:110 and the            amino acid sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ            ID NO:8.    -   The TNF family ligand trimer-containing antigen binding molecule        as described herein before, wherein the antigen binding molecule        comprises        -   (i) a first heavy chain comprising a VH domain comprising an            amino acid sequence of SEQ ID NO:118,        -   (ii) two light chains comprising each a VL domain comprising            an amino acid sequence of SEQ ID NO:119, and        -   (iii) a second heavy chain comprising a VH domain comprising            an amino acid sequence of SEQ ID NO:118 and the amino acid            sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8.    -   The TNF family ligand trimer-containing antigen binding molecule        as described herein before, wherein the antigen binding molecule        comprises        -   (i) a first heavy chain comprising a VH domain comprising an            amino acid sequence of SEQ ID NO:126 or a VH domain            comprising an amino acid sequence of SEQ ID NO:134,        -   (ii) two light chains comprising a VL domain comprising an            amino acid sequence of SEQ ID NO:127 or a VL domain            comprising an amino acid sequence of SEQ ID NO:135, and        -   (iii) a second heavy chain comprising a VH domain comprising            an amino acid sequence of SEQ ID NO:126 or a VH domain            comprising an amino acid sequence of SEQ ID NO:134 and the            amino acid sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ            ID NO:8.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the antigen binding molecule        comprises        -   (a) a heavy chain and a light chain, both comprising a Fab            molecule capable of specific binding to a target cell            antigen, and        -   (b) a fusion protein comprising a Fc domain and a            polypeptide comprising the amino acid sequence of SEQ ID            NO:5.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the antigen binding molecule        comprises        -   (a) a heavy chain and a light chain, both comprising a Fab            molecule capable of specific binding to a target cell            antigen, and        -   (b) the fusion protein comprising the amino acid sequence of            SEQ ID NO:15.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the antigen binding molecule        comprises        -   (i) a variable heavy chain comprising the amino acid            sequence of SEQ ID NO:16,        -   (ii) a variable light chain comprising the amino acid            sequence of SEQ ID NO:17, and        -   (iii) a polypeptide comprising the amino acid sequence of            SEQ ID NO:5.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the antigen binding molecule        comprises        -   (a) a heavy chain and a light chain, both comprising a Fab            molecule capable of specific binding to a target cell            antigen, and        -   (b) a fusion protein comprising a Fc domain and a            polypeptide comprising the amino acid sequence of SEQ ID            NO:8.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the antigen binding molecule        comprises        -   (a) a heavy chain and a light chain, both comprising a Fab            molecule capable of specific binding to a target cell            antigen, and        -   (b) the fusion protein comprising the amino acid sequence of            SEQ ID NO:20.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, wherein the antigen binding molecule        comprises        -   (i) a variable heavy chain comprising the amino acid            sequence of SEQ ID NO:16,        -   (ii) a variable light chain comprising the amino acid            sequence of SEQ ID NO:17, and        -   (iii) a polypeptide comprising the amino acid sequence of            SEQ ID NO:8.    -   An isolated polynucleotide encoding the TNF family ligand        trimer-containing antigen binding molecule as described before.    -   A vector, particularly an expression vector, comprising the        isolated polynucleotide as described before.    -   A host cell comprising the isolated polynucleotide as described        before or the vector as described before.    -   A method for producing the TNF family ligand trimer-containing        antigen binding molecule as described before, comprising the        steps of        -   (i) culturing the host cell of as described before under            conditions suitable for expression of the antigen binding            molecule, and        -   (ii) recovering the antigen binding molecule.    -   A pharmaceutical composition comprising the TNF family ligand        trimer-containing antigen binding molecule as described before        and at least one pharmaceutically acceptable excipient.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, or the pharmaceutical composition as        described before, for use as a medicament.    -   The TNF family ligand trimer-containing antigen binding molecule        as described before, or the pharmaceutical composition as        described before, for use in the treatment of cancer.    -   Use of the TNF family ligand trimer-containing antigen binding        molecule as described before for the manufacture of a medicament        for the treatment of cancer.    -   A method of treating a disease in an individual, comprising        administering to said individual a therapeutically effective        amount of a composition comprising TNF family ligand        trimer-containing antigen binding molecule as described before        in a pharmaceutically acceptable form.    -   The method of treating a disease as described before, wherein        said disease is cancer.

EXAMPLE S

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook etal., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, New York, 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions. General information regarding the nucleotide sequences ofhuman immunoglobulin light and heavy chains is given in: Kabat, E. A. etal., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed.,NIH Publication No 91-3242.

DNA Sequencing

DNA sequences were determined by double strand sequencing.

Gene Synthesis

Desired gene segments were either generated by PCR using appropriatetemplates or were synthesized by Geneart AG (Regensburg, Germany) fromsynthetic oligonucleotides and PCR products by automated gene synthesis.In cases where no exact gene sequence was available, oligonucleotideprimers were designed based on sequences from closest homologues and thegenes were isolated by RT-PCR from RNA originating from the appropriatetissue. The gene segments flanked by singular restriction endonucleasecleavage sites were cloned into standard cloning / sequencing vectors.The plasmid DNA was purified from transformed bacteria and concentrationdetermined by UV spectroscopy. The DNA sequence of the subcloned genefragments was confirmed by DNA sequencing. Gene segments were designedwith suitable restriction sites to allow sub-cloning into the respectiveexpression vectors. All constructs were designed with a 5′-end DNAsequence coding for a leader peptide which targets proteins forsecretion in eukaryotic cells.

Cell Culture Techniques

Standard cell culture techniques were used as described in CurrentProtocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford,J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley &Sons, Inc.

Protein Purification

Proteins were purified from filtered cell culture supernatants referringto standard protocols. In brief, antibodies were applied to a Protein ASepharose column (GE healthcare) and washed with PBS. Elution ofantibodies was achieved at pH 2.8 followed by immediate neutralizationof the sample. Aggregated protein was separated from monomericantibodies by size exclusion chromatography (Superdex 200, GEHealthcare) in PBS or in 20 mM Histidine, 150 mM NaCl pH 6.0. Monomericantibody fractions were pooled, concentrated (if required) using e.g., aMILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozen andstored at −20° C. or −80° C. Part of the samples were provided forsubsequent protein analytics and analytical characterization e.g. bySDS-PAGE, size exclusion chromatography (SEC) or mass spectrometry.

SDS-PAGE

The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to themanufacturer's instruction. In particular, 10% or 4-12% NuPAGE® Novex®Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, withNuPAGE® Antioxidant running buffer additive) or MOPS (non-reduced gels)running buffer was used.

Analytical Size Exclusion Chromatography

Size exclusion chromatography (SEC) for the determination of theaggregation and oligomeric state of antibodies was performed by HPLCchromatography. Briefly, Protein A purified antibodies were applied to aTosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH₂PO₄/K₂HPO₄, pH 7.5on an Agilent HPLC 1100 system or to a Superdex 200 column (GEHealthcare) in 2× PBS on a Dionex HPLC-System. The eluted protein wasquantified by UV absorbance and integration of peak areas. BioRad GelFiltration Standard 151-1901 served as a standard.

Mass Spectrometry

This section describes the characterization of the multispecificantibodies with VH/VL exchange (VH/VL CrossMabs) with emphasis on theircorrect assembly. The expected primary structures were analyzed byelectrospray ionization mass spectrometry (ESI-MS) of the deglycosylatedintact CrossMabs and deglycosylated/plasmin digested or alternativelydeglycosylated/limited LysC digested CrossMabs.

The VH/VL CrossMabs were deglycosylated with N-Glycosidase F in aphosphate or Tris buffer at 37° C. for up to 17 h at a proteinconcentration of 1 mg/ml. The plasmin or limited LysC (Roche) digestionswere performed with 100 μg deglycosylated VH/VL CrossMabs in a Trisbuffer pH 8 at room temperature for 120 hours and at 37° C. for 40 min,respectively. Prior to mass spectrometry the samples were desalted viaHPLC on a Sephadex G25 column (GE Healthcare). The total mass wasdetermined via ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik)equipped with a TriVersa NanoMate source (Advion).

Example 1 Preparation and Purification of Targeted Single Chain 4-1BBLigand Trimer-Containing Fc Fusion Antigen Binding Molecules

1.1. Monovalent FAP(28H1)-Targeted Single Chain 4-1BB LigandTrimer-Containing Fc (kih) Fusion Antigen Binding Molecule (Compound S1)

The DNA sequence encoding part of the ectodomain (amino acids 71-254 or71-248) of human 4-1BB ligand was synthetized according to UniProtAccession No. P41273 (SEQ ID NO:43).

A polypeptide comprising three ectodomains of 4-1BB ligand, eachseparated by a (G₄5)₂ linker (SEQ ID NO:54), was cloned as described inFIG. 1A: human 4-1BB ligand, (G₄5)₂ connector, human 4-1BB ligand,(G₄5)₂ connector, human 4-1BB ligand.

The polypeptide encoding the single chain trimeric 4-1BB ligand wassubcloned in frame with the human IgG1 heavy chain CH2 and CH3 domainson the knob (Merchant, Zhu et al., 1998), using a linker (GSPGSSSSGS) ofSEQ ID NO:57.

The variable region of heavy and light chain DNA sequences encoding abinder specific for fibroblast activation protein (FAP), clone 28H1,were subcloned in frame with either the constant heavy chain of the hole(Carter, 2001) or the constant light chain of human IgG1. The generationand preparation of the FAP binders is described in WO 2012/020006 A2,which is incorporated herein by reference.

The Pro329Gly, Leu234Ala and Leu235Ala mutations were introduced in theconstant region of the knob and hole heavy chains to abrogate binding toFc gamma receptors according to the method described in InternationalPatent Appl. Publ. No. WO 2012/130831 A1.

Combination of the single chain ligand-Fc knob chain containing theS354C/T366W mutations, with the targeted anti-FAP-Fc hole chaincontaining the Y349C/T366S/L368A/Y407V mutations and the anti-FAP lightchain, allows generation of a heterodimer, which includes a single chain4-1BB ligand trimer and a FAP binding Fab (FIGS. 3A-C).

Table 1 shows, respectively, the cDNA and amino acid sequences of theFAP(28H1)-targeted single chain 4-1BB ligand trimer-containing Fc (kih)fusion antigen binding molecule.

TABLE 1 Sequences of FAP(28H1)-targeted human 4-1BB ligandtrimer-containing Fc (kih) fusion molecule (FAP singlechain 4-1BBL trimer) (compound S1) SEQ ID NO: Description Sequence 66nucleotide AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGG sequence ofACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGT trimeric hu 4-1BBLGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTG (71-254)-Fc knobGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGG chainCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCTCCAAGAAGCGAAGGCGGAGGCGGATCTGGCGGCGGAGGATCTAGAGAGGGACCCGAACTGTCCCCTGACGATCCAGCCGGGCTGCTGGATCTGAGACAGGGAATGTTCGCCCAGCTGGTGGCTCAGAATGTGCTGCTGATTGACGGACCTCTGAGCTGGTACTCCGACCCAGGGCTGGCAGGGGTGTCCCTGACTGGGGGACTGTCCTACAAAGAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAGGGCTCAGGATCTGTGTCCCTGGCTCTGCATCTGCAGCCACTGCGCTCTGCTGCTGGCGCAGCTGCACTGGCTCTGACTGTGGACCTGCCACCAGCCTCTAGCGAGGCCAGAAACAGCGCCTTCGGGTTCCAAGGACGCCTGCTGCATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCATACTGAAGCCAGAGCCCGGCATGCTTGGCAGCTGACTCAGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACACCTGAGATCCCTGCCGGACTGCCAAGCCCTAGATCAGAAGGGGGCGGAGGAAGCGGAGGGGGAGGAAGTAGAGAAGGACCAGAGCTGTCTCCAGATGACCCCGCAGGACTGCTGGACCTGAGACAGGGCATGTTCGCACAGCTGGTGGCCCAGAATGTGCTGCTGATCGACGGGCCACTGTCTTGGTATTCCGATCCCGGCCTGGCCGGCGTGTCCCTGACCGGCGGACTGAGTTACAAAGAGGATACAAAAGAACTGGTGGTGGCAAAGGCAGGGGTGTACTATGTGTTCTTTCAGCTGGAACTGAGAAGGGTGGTGGCCGGCGAGGGAAGCGGATCAGTGTCACTGGCACTGCATCTGCAGCCCCTGAGATCCGCTGCAGGGGCCGCTGCTCTGGCCCTGACCGTGGACCTGCCCCCTGCTTCTTCCGAGGCTAGAAACTCTGCATTTGGGTTTCAAGGACGCCTGCTGCATCTGTCAGCCGGGCAGAGACTGGGAGTGCATCTGCATACCGAGGCTCGCGCCAGACATGCATGGCAGCTGACCCAGGGCGCCACCGTGCTGGGACTGTTTAGAGTGACTCCAGAAATCCCCGCTGGCCTGCCCAGCCCAAGATCCGAGGGATCTCCTGGCAGCAGCTCTAGCGGATCCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC CGGGTAAA 67 anti-FAP (28H1)GAAGTGCAGCTGCTGGAATCCGGCGGAGGCCTGGTGCA Fc hole chainGCCTGGCGGATCTCTGAGACTGTCCTGCGCCGCCTCCGGCTTCACCTTCTCCTCCCACGCCATGTCCTGGGTCCGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGTCCGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGACTCTGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGGCTGGCTGGGCAACTTCGACTACTGGGGACAGGGCACCCTGGTCACCGTGTCCAGCGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC GCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA68 anti-FAP(28H1) GAGATCGTGCTGACCCAGTCCCCCGGCACCCTGTCTCTG light chainAGCCCTGGCGAGAGAGCCACCCTGTCCTGCAGAGCCTCCCAGTCCGTGTCCCGGTCCTACCTCGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCTCGGCTGCTGATCATCGGCGCCTCTACCAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCCAGGTCATCCCTCCCACCTTTGGCCAGGGCACCAAGGTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCA CAAAGAGCTTCAACAGGGGAGAGTGT 15trimeric hu 4- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWY 1BBL (71-254)-SDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELR Fc knob chainRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGSPGSSSSGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK 18 anti-FAP(28H1)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQA Fc hole chainPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 19 anti-FAP (28H1)EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKP light chainGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC

The targeted single chain TNF ligand trimer-containing Fc (kih) fusionantigen binding molecule encoding sequences were cloned into a plasmidvector driving expression of the insert from an MPSV promoter andcontaining a synthetic polyA sequence located at the 3′ end of the CDS.In addition, the vector contained an EBV OriP sequence for episomalmaintenance of the plasmid.

The targeted single chain 4-1BBL-containing Fc (kih) fusion antigenbinding molecule was produced by co-transfecting HEK293-EBNA cells withthe mammalian expression vectors using polyethylenimine. The cells weretransfected with the corresponding expression vectors at a 1:1:1 ratio(“vector knob chain”: “vector hole chain”: “vector light chain”).

For production in 500 mL shake flasks, 400 million HEK293 EBNA cellswere seeded 24 hours before transfection. For transfection cells werecentrifuged for 5 minutes at 210×g, and the supernatant was replaced bypre-warmed CD CHO medium. Expression vectors were mixed in 20 mL CD CHOmedium to a final amount of 200 μg DNA. After addition of 540 μL PEI,the solution was vortexed for 15 seconds and incubated for 10 minutes atroom temperature.

Afterwards, cells were mixed with the DNA/PEI solution, transferred to a500 mL shake flask and incubated for 3 hours at 37° C. in an incubatorwith a 5% CO₂ atmosphere. After the incubation, 160 mL of F17 medium wasadded and cells were cultured for 24 hours. One day after transfection 1mM valproic acid and 7% Feed with supplements were added. Afterculturing for 7 days, the supernatant was collected by centrifugationfor 15 minutes at 210×g. The solution was sterile filtered (0.22 μmfilter), supplemented with sodium azide to a final concentration of0.01% (w/v), and kept at 4° C.

The targeted 4-1BBL trimer-containing Fc (kih) fusion antigen bindingmolecule was purified from cell culture supernatants by affinitychromatography using Protein A, followed by size exclusionchromatography. For affinity chromatography, the supernatant was loadedon a

HiTrap ProteinA HP column (CV=5 mL, GE Healthcare) and equilibrated with20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride,0.01% (v/v) Tween-20 containing buffer (pH 7.5). Unbound protein wasremoved by washing with at least 10 column volumes of the same buffer.The bound protein was eluted using a linear pH gradient over 20 columnvolumes to 100% of 20 mM sodium citrate, 0.5 M sodium chloride, 0.01%(v/v) Tween-20 containing buffer (pH 2.5). The column was then washedwith 10 column volumes of 20 mM sodium citrate, 0.5 M sodium chloride,0.01% Tween-20 buffer (pH 2.5).

The pH of the collected fractions was adjusted by adding 1/40 (v/v) of2M Tris, pH8.0. The protein was concentrated prior to loading on aHiLoad Superdex 200 column (GE Healthcare) equilibrated with 20 mMHistidine, 150 mM sodium chloride, 0.01% (v/v) Tween/20 solution of pH6.0.

The protein concentration was determined by measuring the opticaldensity (OD) at 280 nm, using a molar extinction coefficient calculatedon the basis of the amino acid sequence. Purity and molecular weight ofthe targeted TNF ligand trimer-containing Fc (kih) fusion antigenbinding molecule was analyzed by SDS-PAGE in the presence and absence ofa reducing agent (5 mM 1,4-dithiotreitol) and staining with CoomassieSimpleBlueTM SafeStain (Invitrogen USA). The aggregate content ofsamples was analyzed using a TSKgel G3000 SW XL analyticalsize-exclusion column (Tosoh) equilibrated in 25 mM K₂HPO₄, 125 mM NaCl,200 mM L-Arginine Monohydrocloride, 0.02% (w/v) NaN₃, pH 6.7 runningbuffer at 25° C.

Table 2 summarizes the yield and final monomer content of theFAP-targeted 4-1BBL trimer-containing Fc (kih) fusion antigen bindingmolecule.

TABLE 2 Summary of the production of the FAP-targeted 4-1BBLtrimer-containing Fc (kih) fusion antigen binding molecule Monomer Yield[%] LC/MS Construct [mg/l] (SEC) (non red) FAP-targeted 4-1BBL trimer-14 100 Theoretical*: containing Fc (kih) fusion 157358.6 Da antigenbinding molecule Experimental: (FAP-targeted single chain 157384 Da4-1BBL trimer) *without Compound S1 terminal lysines

1.2. Bivalent FAP(4B9)-Targeted Single Chain 4-1BB LigandTrimer-Containing Fc (kih) Fusion Antigen Binding Molecule (Compound S2)

A polypeptide containing three ectodomains of 4-1BB ligand (71-254 or71-248, synthetized according to P41273), separated by (G45)₂ linkerswas subcloned in frame at the C-terminus of human IgG1 Fc knob chain(Merchant, Zhu et al., 1998), as depicted in FIG. 28A: human IgG1 Fcknob, (G45)2 connector, human 4-1BB ligand, (G4S)2 connector, human4-1BB ligand, (G4S)2 connector, human 4-1BB ligand.

The variable region of heavy and light chain DNA sequences encoding abinder specific for fibroblast activation protein (FAP), clone 4B9, weresubcloned in frame with either the constant heavy chain of the hole, theknob or the constant light chain of human IgG1. The generation andpreparation of the FAP binders is described in WO 2012/020006 A2, whichis incorporated herein by reference.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have been introduced inthe constant region of the knob and hole heavy chains to abrogatebinding to Fc gamma receptors as described in WO 2012/130831 A1.

Combination of the anti-FAP huIgG1 hole chain containing theY349C/T366S/L368A/Y407Vmutations, the anti-FAP huIgG1 knob trimericligand chain containing the

S354C/T366W mutations and the anti-FAP light chain allows generation ofa heterodimer, which includes an assembled trimeric 4-1BB ligand and twoFAP binding Fabs (FIG. 28B).

Table 3 shows, respectively, the cDNA and amino acid sequences of thebivalent FAP-targeted single chain 4-1BB ligand trimer-containing Fc(kih) fusion antigen binding molecule.

TABLE 3Sequences of bivalent FAP(4B9)-targeted single chain 4-1BBL(71-254)trimer-containing Fc (kih) fusion antigen binding molecule (Compound S2)SEQ ID NO: Description Sequence 137 nucleotideGAGGTGCAGCTGCTCGAAAGCGGCGGAGGACTGGTGCA sequence of anti-GCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCG FAP(4B9) FcGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCC hole chainAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATCATCGGCTCTGGCGCCAGCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAAGGGATGGTTCGGCGGCTTCAACTACTGGGGACAGGGCACCCTGGTCACAGTGTCCAGCGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 138 nucleotideGAGGTGCAGCTGCTCGAAAGCGGCGGAGGACTGGTGCA sequence of anti-GCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCG FAP(4B9) FcGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCC knob chain fusedAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATC to trimeric hu 4-ATCGGCTCTGGCGCCAGCACCTACTACGCCGACAGCGTG 1BBL (71-254)AAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAAGGGATGGTTCGGCGGCTTCAACTACTGGGGACAGGGCACCCTGGTCACAGTGTCCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGCCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGCGGAGGCGGAGGATCTGGCGGGGGAGGATCTAGAGAGGGCCCTGAGCTGTCCCCCGATGATCCTGCTGGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGCTGATCGACGGCCCTCTGAGCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAGGGATCTGGATCTGTGTCTCTGGCCCTGCATCTGCAGCCCCTGAGATCAGCTGCTGGCGCTGCTGCTCTGGCTCTGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAAGCCAGAGCCAGGCACGCTTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCTCCAAGAAGCGAAGGCGGCGGAGGCTCCGGAGGAGGCGGCTCTAGAGAAGGACCTGAACTGAGCCCAGACGACCCCGCAGGGCTGCTGGATCTGAGACAGGGAATGTTCGCCCAGCTGGTGGCTCAGAATGTGCTGCTGATTGATGGACCCCTGTCCTGGTACTCCGACCCAGGGCTGGCAGGGGTGTCCCTGACTGGCGGACTGTCTTACAAAGAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAAGGCAGTGGCTCTGTGTCCCTGGCACTGCATCTGCAGCCTCTGCGCAGTGCTGCAGGCGCAGCCGCACTGGCACTGACTGTGGACCTGCCCCCAGCTTCCAGCGAGGCTAGAAACAGCGCCTTCGGGTTTCAAGGACGCCTGCTGCATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCATACTGAGGCTAGGGCCAGACATGCCTGGCAGCTGACCCAGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACACCTGAGATCCCCGCTGGCCTGCCAAGCCCTAGATCAGAAGGCGGAGGGGGATCAGGGGGAGGCGGATCCAGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCTCC AAGAAGCGAA 139 nucleotideGAGATCGTGCTGACCCAGTCCCCCGGCACCCTGTCTCTG sequence of anti-AGCCCTGGCGAGAGAGCCACCCTGTCCTGCAGAGCCTC FAP(4B9) lightCCAGTCCGTGACCTCCTCCTACCTCGCCTGGTATCAGCA chainGAAGCCCGGCCAGGCCCCTCGGCTGCTGATCAACGTGGGCAGTCGGAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCATCATGCTGCCCCCCACCTTTGGCCAGGGCACCAAGGTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCA CAAAGAGCTTCAACAGGGGAGAGTGT 140anti-FAP(4B9) Fe EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA hole chainPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 141 anti-FAP(4B9) FcEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA knob chain fusedPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYL to trimeric 4 huQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSAS 4-1BBL (71-254)TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHA WQLTQGATVLGLFRVTPEIPAGLPSPRSE142 anti-FAP(4B9) EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKP light chainGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC

1.3. Monovalent FAP(4B9)-Targeted Single Chain 4-1BB LigandTrimer-Containing Fc (kih) Fusion Antigen Binding Molecule (Compound S3)

A polypeptide containing three ectodomains of 4-1BB ligand (71-254 or71-248, synthetized according to P41273), separated by (G4S)₂ linkerswas subcloned as depicted in FIG. 1A: human 4-1BB ligand, (G4S)2connector, human 4-1BB ligand, (G4S)2 connector, human 4-1BB ligand.

The polypeptide encoding the single chain trimeric 4-1BB ligand wassubcloned in frame with the human IgG1 heavy chain CH2 and CH3 domainson the knob (Merchant, Zhu et al, 1998), using a linker (GSPGSSSSGS) ofSEQ ID NO:57.

The variable region of heavy and light chain DNA sequences encoding abinder specific for fibroblast activation protein (FAP), clone 4B9, weresubcloned in frame with either the constant heavy chain of the hole, theknob or the constant light chain of human IgG1. The generation andpreparation of the FAP binder is described in WO 2012/020006 A2, whichis incorporated herein by reference.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have been introduced inthe constant region of the knob and hole heavy chains to abrogatebinding to Fc gamma receptors as described in WO 2012/130831 A1.

Combination of the single chain ligand-Fc knob chain containing theS354C/T366W mutations, with the targeted anti-FAP-Fc hole chaincontaining the Y349C/T366S/L368A/Y407V mutations and the anti-FAP lightchain, allows generation of a heterodimer, which includes a single chain4-1BB ligand trimer and a FAP binding Fab (FIG. 3A).

Table 4 shows, respectively, the cDNA and amino acid sequences of themonovalent FAP(4B9)-targeted single chain 4-1BB ligand trimer-containingFc (kih) fusion antigen binding molecule.

TABLE 4 Sequences of monovalent FAP(4B9)-targeted single chain4-1BBL(71-254) trimer containing Fc (kih) fusion antigen bindingmolecule (Compound S3) SEQ ID NO: Description Sequence 137 nucleotidesequence of anti- see Table 3 FAP(4B9) Fc hole chain 66 nucleotidesequence of trimeric see Table 1 hu 4-1BBL (71-254) Fc knob chain 139nucleotide sequence of anti- see Table 3 FAP(4B9) light chain 140anti-FAP(4B9) Fc hole chain see Table 3 15 trimeric hu 4-1BBL (71-254)see Table 1 Fc knob chain 142 anti-FAP(4B9) light chain see Table 3

1.4. Bivalent CEA-Targeted Single Chain 4-1BB Ligand Trimer-ContainingFc (kih) Fusion Antigen Binding Molecule (Compound S4)

The molecule was prepared as described in 1.2 for the bivalentFAP(4B9)-targeted construct, with the only difference that the anti-FAPbinder was replaced by an anti-CEA binder.

The variable region of heavy and light chain DNA sequences encoding abinder specific for carcinoembryonic antigen (CEA), clone sm9b, weresubcloned in frame with either the constant heavy chain of the hole, theknob or the constant light chain of human IgG1. The clone sm9b isfurther described in US 2005/0147614, Graff et al. 2004 and WO2014/004694 A1 which are incorporated herein by reference.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have been introduced inthe constant region of the knob and hole heavy chains to abrogatebinding to Fc gamma receptors as described in WO 2012/130831 A1.

Combination of the anti-CEA huIgG1 hole chain containing theY349C/T366S/L368A/Y407Vmutations, the anti-CEA huIgG1 knob trimericligand chain containing the S354C/T366W mutations and the anti-CEA lightchain allows generation of a heterodimer, which includes an assembledtrimeric 4-1BB ligand and two CEA binding Fabs (FIG. 28B).

Table 5 shows, respectively, the cDNA and amino acid sequences of thebivalent CEA(sm9b)-targeted single chain 4-1BB ligand trimer-containingFc (kih) fusion antigen binding molecule.

TABLE 5 Sequences of bivalent CEA(sm9b)-targeted single chain 4-1BBL(71-254) trimer-containing Fc (kih) fusion antigen bindingmolecule (Compound S4) SEQ ID NO: Description Sequence 143 nucleotideCAAGTGAAGCTGGAGCAGAGCGGCGCCGAAGTCGTGAA sequence of anti-ACCCGGGGCTTCCGTCAAGCTCTCTTGCAAGGCATCAGG CEA(sm9b) FcATTCAACATCAAAGACAGCTACATGCACTGGCTGAGGC hole chainAGGGCCCTGGTCAGTGCCTTGAGTGGATTGGCTGGATCGATCCAGAGAATGGCGACACCGAATATGCCCCCAAGTTTCAAGGAAAGGCTACATTCACCACTGATACATCCGCAAACACCGCCTACCTGGGTCTCTCAAGTCTGCGCCCTGAGGACACTGCTGTGTATTACTGTAATGAGGGCACCCCAACAGGGCCCTACTATTTTGACTACTGGGGACAGGGCACCTTGGTTACAGTGAGCTCCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT AAA 144 nucleotideCAAGTGAAGCTGGAGCAGAGCGGCGCCGAAGTCGTGAA sequence of anti-ACCCGGGGCTTCCGTCAAGCTCTCTTGCAAGGCATCAGG CEA(sm9b) FcATTCAACATCAAAGACAGCTACATGCACTGGCTGAGGC knob chain fusedAGGGCCCTGGTCAGTGCCTTGAGTGGATTGGCTGGATCG to trimeric hu 4-ATCCAGAGAATGGCGACACCGAATATGCCCCCAAGTTT 1BBL (71-254)CAAGGAAAGGCTACATTCACCACTGATACATCCGCAAACACCGCCTACCTGGGTCTCTCAAGTCTGCGCCCTGAGGACACTGCTGTGTATTACTGTAATGAGGGCACCCCAACAGGGCCCTACTATTTTGACTACTGGGGACAGGGCACCTTGGTTACAGTGAGCTCCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGCCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGCGGAGGCGGAGGATCTGGCGGGGGAGGATCTAGAGAGGGCCCTGAGCTGTCCCCCGATGATCCTGCTGGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGCTGATCGACGGCCCTCTGAGCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAGGGATCTGGATCTGTGTCTCTGGCCCTGCATCTGCAGCCCCTGAGATCAGCTGCTGGCGCTGCTGCTCTGGCTCTGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAAGCCAGAGCCAGGCACGCTTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCTCCAAGAAGCGAAGGCGGCGGAGGCTCCGGAGGAGGCGGCTCTAGAGAAGGACCTGAACTGAGCCCAGACGACCCCGCAGGGCTGCTGGATCTGAGACAGGGAATGTTCGCCCAGCTGGTGGCTCAGAATGTGCTGCTGATTGATGGACCCCTGTCCTGGTACTCCGACCCAGGGCTGGCAGGGGTGTCCCTGACTGGCGGACTGTCTTACAAAGAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAAGGCAGTGGCTCTGTGTCCCTGGCACTGCATCTGCAGCCTCTGCGCAGTGCTGCAGGCGCAGCCGCACTGGCACTGACTGTGGACCTGCCCCCAGCTTCCAGCGAGGCTAGAAACAGCGCCTTCGGGTTTCAAGGACGCCTGCTGCATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCATACTGAGGCTAGGGCCAGACATGCCTGGCAGCTGACCCAGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACACCTGAGATCCCCGCTGGCCTGCCAAGCCCTAGATCAGAAGGCGGAGGGGGATCAGGGGGAGGCGGATCCAGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCT CCAAGAAGCGAA 145 nucleotideGAGAACGTGCTGACCCAGAGCCCCTCCTCTATGTCAGTC sequence of anti-AGCGTGGGCGACAGGGTCACAATCGCCTGCTCCGCTTCT CEA(sm9b) lightAGTAGCGTGCCTTACATGCACTGGCTCCAGCAGAAGCC chainAGGGAAATCCCCCAAGCTGCTTATTTATTCTACCTCAAATCTGGCAAGCGGAGTTCCTAGCAGATTCTCTGGCAGTGGTAGCGGGACTGATTACTCCCTCACAATCTCAAGTGTGCAGCCAGAAGACGCCGCTACCTATTACTGTCAACAGCGCAGCTCCTACCCCCTGACTTTTGGCTGTGGCACCAAGTTGGAGATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAA GAGCTTCAACAGGGGAGAGTGT 146anti-CEA(sm9b) QVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQ Fc hole chainGPGQCLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 147 anti-CEA(sm9b)QVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQ Fc knob chainGPGQCLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTA fused to trimericYLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVS hu 4-1BBL (71-SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS 254)WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 148 anti-CEA(sm9b)ENVLTQSPSSMSVSVGDRVTIACSASSSVPYMHWLQQKPG light chainKSPKLLIYSTSNLASGVPSRFSGSGSGTDYSLTISSVQPEDAATYYCQQRSSYPLTFGCGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC

1.5. Monovalent CEA(sm9b)-Targeted Single Chain 4-1BB LigandTrimer-C5ontaining Fc (kih) Fusion Antigen Binding Molecule (CompoundS5)

The molecule was prepared as described in 1.3 for the monovalentFAP-targeted construct, with the only difference that the anti-FAPbinder was replaced by an anti-CEA binder.

The variable region of heavy and light chain DNA sequences encoding abinder specific for fibroblast activation protein (CEA), clone sm9b,were subcloned in frame with either the constant heavy chain of the hole(Carter, 2001) or the constant light chain of human IgG1.

Combination of the anti-CEA huIgG1 hole chain containing theY349C/T366S/L368A/Y407Vmutations, the single chain ligand huIgG1 knobchain containing the S354C/T366W mutations and the anti-CEA light chainallows generation of a heterodimer, which includes an assembled trimeric4-1BB ligand and one CEA binding Fab (FIG. 2A).

Table 6 shows, respectively, the cDNA and amino acid sequences of themonovalent CEA(sm9b)-targeted single chain 4-1BB ligandtrimer-containing Fc (kih) fusion antigen binding molecule.

TABLE 6 Sequences of monovalent CEA(sm9b)- targeted single chain4-1BBL(71-254) trimer-containing Fc (kih) fusion antigen bindingmolecule (Compound S5) SEQ ID NO: Description Sequence 143 nucleotidesequence of anti- see Table 5 CEA(sm9b) Fc hole chain 66 nucleotidesequence of trimeric see Table 1 hu 4-1BBL (71-254) Fc knob chain 145nucleotide sequence of anti- see Table 5 CEA(sm9b) light chain 146anti-CEA(sm9b) Fc hole chain see Table 5 15 trimeric hu 4-1BBL (71-254)Fc see Table 1 knob chain 148 anti-CEA(sm9b) light chain see Table 5

1.6. Bivalent CD19-targeted single chain 4-1BB ligand trimer-containingFc (kih) fusion antigen binding molecules (Compound S6 and S7)Themolecules are prepared as described in 1.2 for the bivalentFAP(4B9)-targeted construct, with the only difference that the anti-FAPbinder is replaced by anti-CD19 binders. The generation of the CD19clones is described in Example 1.8.

The variable region of heavy and light chain DNA sequences encoding abinder specific for CD19, i.e. clones 8B8-018 and 8B8-2B11, aresubcloned in frame with either the constant heavy chain of the hole, theknob or the constant light chain of human IgG1.

Combination of the anti-CD19 hu IgG1 hole chain containing theY349C/T366S/L368A/Y407Vmutations, the anti-CD19 hu IgG1 knob trimericligand chain containing the S354C/T366W mutations and the anti-CD19light chain allows generation of a heterodimer, which includes anassembled trimeric 4-1BB ligand and two CD19 binding Fabs (FIG. 28B).The Pro329Gly, Leu234Ala and Leu235Ala mutations are introduced in theconstant region of the knob and hole heavy chains to abrogate binding toFc gamma receptors as described in WO 2012/130831 A1.

Table 7 shows, respectively, the cDNA and amino acid sequences of thebivalent CD19(8B8-018)-targeted single chain 4-1BB ligandtrimer-containing Fc (kih) fusion antigen binding molecule.

TABLE 7 Sequences of bivalent CD19(8B8-018)-targeted single chain 4-1BBL(71-254) trimer-containing Fc (kih) fusion antigen bindingmolecule (Compound S6) SEQ ID NO: Description Sequence 149 nucleotideCAGGTCCAGCTGGTGCAGTCCGGCGCCGAGGTCAAGAA sequence of anti-ACCCGGGGCTTCTGTGAAGGTTTCATGCAAGGCAAGCG CD19(8B8-018)GATACACCTTCACCGACTATATCATGCATTGGGTCAGGC Fc hole chainAGGCCCCTGGCCAAGGTCTCGAATGGATGGGCTACATTAACCCATATAATGATGGCTCCAAATACACCGAGAAGTTTCAGGGAAGAGTCACTATGACATCTGACACCAGTATCAGCACTGCTTACATGGAGCTGTCCCGCCTTCGGTCTGATGACACCGCAGTGTATTACTGTGCCAGGGGCACATATTACTACGGCTCAGCTCTGTTCGACTATTGGGGGCAGGGAACCACAGTAACCGTGAGCTCCGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAA 150 nucleotideCAGGTCCAGCTGGTGCAGTCCGGCGCCGAGGTCAAGAA sequence of anti-ACCCGGGGCTTCTGTGAAGGTTTCATGCAAGGCAAGCG CD19(8B8-018)GATACACCTTCACCGACTATATCATGCATTGGGTCAGGC Fc knob chainAGGCCCCTGGCCAAGGTCTCGAATGGATGGGCTACATTA fused to trimericACCCATATAATGATGGCTCCAAATACACCGAGAAGTTTC hu 4-1BBL (71-AGGGAAGAGTCACTATGACATCTGACACCAGTATCAGC 254)ACTGCTTACATGGAGCTGTCCCGCCTTCGGTCTGATGACACCGCAGTGTATTACTGTGCCAGGGGCACATATTACTACGGCTCAGCTCTGTTCGACTATTGGGGGCAGGGAACCACAGTAACCGTGAGCTCCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGCCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGCGGAGGCGGAGGATCTGGCGGGGGAGGATCTAGAGAGGGCCCTGAGCTGTCCCCCGATGATCCTGCTGGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGCTGATCGACGGCCCTCTGAGCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAGGGATCTGGATCTGTGTCTCTGGCCCTGCATCTGCAGCCCCTGAGATCAGCTGCTGGCGCTGCTGCTCTGGCTCTGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAAGCCAGAGCCAGGCACGCTTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCTCCAAGAAGCGAAGGCGGCGGAGGCTCCGGAGGAGGCGGCTCTAGAGAAGGACCTGAACTGAGCCCAGACGACCCCGCAGGGCTGCTGGATCTGAGACAGGGAATGTTCGCCCAGCTGGTGGCTCAGAATGTGCTGCTGATTGATGGACCCCTGTCCTGGTACTCCGACCCAGGGCTGGCAGGGGTGTCCCTGACTGGCGGACTGTCTTACAAAGAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAAGGCAGTGGCTCTGTGTCCCTGGCACTGCATCTGCAGCCTCTGCGCAGTGCTGCAGGCGCAGCCGCACTGGCACTGACTGTGGACCTGCCCCCAGCTTCCAGCGAGGCTAGAAACAGCGCCTTCGGGTTTCAAGGACGCCTGCTGCATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCATACTGAGGCTAGGGCCAGACATGCCTGGCAGCTGACCCAGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACACCTGAGATCCCCGCTGGCCTGCCAAGCCCTAGATCAGAAGGCGGAGGGGGATCAGGGGGAGGCGGATCCAGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCC TTCTCCAAGAAGCGAA 151 nucleotideGACATCGTCATGACCCAGACACCCCTGTCCCTCTCTGTG sequence of anti-ACCCCTGGCCAGCCAGCCTCAATTAGCTGCAAGTCCTCT CD19(8B8-018)CAAAGTCTGGAGAACCCCAATGGGAACACTTACCTTAAT light chainTGGTATCTGCAGAAACCCGGACAATCCCCTCAACTCCTGATCTACAGGGTCTCTAAGAGATTCTCAGGCGTGCCAGATCGCTTTAGCGGTTCCGGGTCTGGCACAGACTTCACCTTGAAGATTAGTCGGGTTGAAGCTGAGGATGTGGGAGTCTATTACTGTCTGCAGCTCACTCATGTGCCCTACACCTTTGGTCAGGGCACAAAACTGGAGATCAAGCGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCCTCCGACGAGCAGCTGAAGTCCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGGGCGAGTGC 152 anti- CD19(8B8-QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ 018) Fc holeAPGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTA chainYMELSRLRSDDTAVYYCARGTYYYGSALFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 153 anti- CD19(8B8-QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ 018) Fc knobAPGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTA chain fused toYMELSRLRSDDTAVYYCARGTYYYGSALFDYWGQGTTVT trimeric hu 4-VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT 1BBL (71-254)VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 154 anti- CD19(8B8-DIVMTQTPLSLSVTPGQPASISCKSSQSLENPNGNTYLNWY 018) light chainLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQLTHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC

Table 8 shows, respectively, the cDNA and amino acid sequences of thebivalent CD19(8B8-2B11)-targeted single chain 4-1BB ligandtrimer-containing Fc (kih) fusion antigen binding molecule.

TABLE 8 Sequences of bivalent CD19(8B8-2B11)-targeted single chain 4-)1BBL(71-254 trimer-containing Fc (kih) fusion antigen bindingmolecule (Compound S7) SEQ ID NO: Description Sequence 155 nucleotideCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAA sequence of anti-ACCGGGCGCTTCCGTTAAAGTGAGCTGCAAAGCATCTGG CD19(8B8-2B11)TTACACCTTCACTGACTATATCATGCACTGGGTTCGTCA Fc hole chainGGCCCCGGGCCAGGGTCTGGAGTGGATGGGCTACATTAACCCATACAACGACGGTTCCAAATATACCGAGAAATTCCAGGGCCGCGTCACGATGACCAGCGACACTTCTATCTCCACCGCGTACATGGAACTGTCTAGACTGCGTTCTGACGACACCGCTGTTTACTATTGTGCACGCGGTACCTACTACTACGGTCCACAGCTGTTTGATTACTGGGGCCAAGGTACCACGGTGACCGTAAGCTCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAA 156 nucleotideCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAA sequence of anti-ACCGGGCGCTTCCGTTAAAGTGAGCTGCAAAGCATCTGG CD19(8B8-2B11)TTACACCTTCACTGACTATATCATGCACTGGGTTCGTCA Fc knob chainGGCCCCGGGCCAGGGTCTGGAGTGGATGGGCTACATTA fused to trimericACCCATACAACGACGGTTCCAAATATACCGAGAAATTC hu 4-1BBL (71-CAGGGCCGCGTCACGATGACCAGCGACACTTCTATCTCC 254)ACCGCGTACATGGAACTGTCTAGACTGCGTTCTGACGACACCGCTGTTTACTATTGTGCACGCGGTACCTACTACTACGGTCCACAGCTGTTTGATTACTGGGGCCAAGGTACCACGGTGACCGTAAGCTCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGCCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGCGGAGGCGGAGGATCTGGCGGGGGAGGATCTAGAGAGGGCCCTGAGCTGTCCCCCGATGATCCTGCTGGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGCTGATCGACGGCCCTCTGAGCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAGGGATCTGGATCTGTGTCTCTGGCCCTGCATCTGCAGCCCCTGAGATCAGCTGCTGGCGCTGCTGCTCTGGCTCTGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAAGCCAGAGCCAGGCACGCTTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCTCCAAGAAGCGAAGGCGGCGGAGGCTCCGGAGGAGGCGGCTCTAGAGAAGGACCTGAACTGAGCCCAGACGACCCCGCAGGGCTGCTGGATCTGAGACAGGGAATGTTCGCCCAGCTGGTGGCTCAGAATGTGCTGCTGATTGATGGACCCCTGTCCTGGTACTCCGACCCAGGGCTGGCAGGGGTGTCCCTGACTGGCGGACTGTCTTACAAAGAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAAGGCAGTGGCTCTGTGTCCCTGGCACTGCATCTGCAGCCTCTGCGCAGTGCTGCAGGCGCAGCCGCACTGGCACTGACTGTGGACCTGCCCCCAGCTTCCAGCGAGGCTAGAAACAGCGCCTTCGGGTTTCAAGGACGCCTGCTGCATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCATACTGAGGCTAGGGCCAGACATGCCTGGCAGCTGACCCAGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACACCTGAGATCCCCGCTGGCCTGCCAAGCCCTAGATCAGAAGGCGGAGGGGGATCAGGGGGAGGCGGATCCAGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCC TTCTCCAAGAAGCGAA 157 nucleotideGATATTGTCATGACTCAAACTCCACTGTCTCTGTCCGTG sequence of anti-ACCCCGGGTCAGCCAGCGAGCATTTCTTGCAAATCCAGC CD19(8B8-2B11)CAATCTCTGGAAACCTCCACCGGCACCACGTACCTGAAC light chainTGGTATCTCCAGAAACCGGGTCAGAGCCCGCAGCTGCTGATCTACCGTGTATCTAAGCGCTTCTCCGGCGTTCCTGATCGTTTCAGCGGTTCTGGATCCGGCACCGACTTTACTCTGAAAATCAGCCGTGTGGAAGCTGAAGACGTTGGCGTCTACTATTGTCTGCAGCTGCTGGAAGATCCATACACCTTCGGTCAAGGAACGAAACTGGAAATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG TGT 158 anti -CD19(8B8-QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ 2B11) Fc holeAPGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTA chainYMELSRLRSDDTAVYYCARGTYYYGPQLFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 159 anti- CD19(8B8-QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ 2B11) Fc knobAPGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTA chain fused toYMELSRLRSDDTAVYYCARGTYYYGPQLFDYWGQGTTVT trimeric hu 4-VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT 1BBL (71-254)VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 160 anti- CD19(8B8-DIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGTTYLNWYL 2B11) light chainQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQLLEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC

1.7. Monovalent CD19-Targeted Single Chain 4-1BB LigandTrimer-Containing Fc (kih) Fusion Antigen Binding Molecules (Compound S8and S9)

The molecules are prepared as described in 1.3 for the monovalentFAP-targeted construct, with the only difference that the anti-FAPbinder is replaced by an anti-CD19 binder.

The variable region of heavy and light chain DNA sequences encoding abinder specific for CD19, i.e. clones 8B8-018 or 8B8-2B11, are subclonedin frame with either the constant heavy chain of the hole (Carter, 2001)or the constant light chain of human IgG1.

Combination of the anti-CD19 hu IgG1 hole chain containing theY349C/T366S/L368A/Y407Vmutations, the single chain ligand hu IgG1 knobchain containing the

S354C/T366W mutations and the anti-CD19 light chain allows generation ofa heterodimer, which includes an assembled trimeric 4-1BB ligand and oneCD19 binding Fab (FIG. 2A).

Table 9 shows, respectively, the cDNA and amino acid sequences of themonovalent CD19(8B8-018)-targeted single chain 4-1BB ligandtrimer-containing Fc (kih) fusion antigen binding molecule.

TABLE 9 Sequences of monovalent CD19(8B8-018)- targeted single chain4-1BBL(71-254) trimer-containing Fc (kih) fusion antigen bindingmolecule (Compound S8) SEQ ID NO: Description Sequence 149 nucleotidesequence of anti- see Table 7 CD19(8B8-018) Fc hole chain 66 nucleotidesequence of trimeric see Table 1 hu 4-1BBL (71-254) Fc knob chain 151nucleotide sequence of anti- see Table 7 CD19(8B8-018) light chain 152anti-CD19(8B8-018) Fc see Table 7 hole chain 15 trimeric hu 4-1BBL(71-254) see Table 1 Fc knob chain 154 anti-CD19(8B8-018) light chainsee Table 7

Table 10 shows, respectively, the cDNA and amino acid sequences of themonovalent CD19(8B8-2B11)-targeted single chain 4-1BB ligandtrimer-containing Fc (kih) fusion antigen binding molecule.

TABLE 10 Sequences of monovalent CD19(8B8-2B11)- targeted single chain4-1BBL(71-254) trimer-containing Fc (kih) fusion antigen bindingmolecule (Compound S9) SEQ ID NO: Description Sequence 155 nucleotidesequence of anti- see Table 8 CD19(8B8-2B11) Fc hole chain 66 nucleotidesequence of trimeric see Table 1 hu 4-1BBL (71-254) Fc knob chain 157nucleotide sequence of anti- see Table 8 CD19(8B8-2B11) light chain 158anti-CD19(8B8-2B11) Fc hole see Table 8 chain 15 trimeric hu 4-1BBL(71-254) Fc see Table 1 knob chain 160 anti-CD19(8B8-2B11) light chainsee Table 8

1.8. Generation of Anti-CD19 Binders 8B8-018 and 8B8-2B11

1.8.1 Generation of Anti-CD19 Clone 8B8-018

a) Immunization and Generation of Mouse Anti-Human CD19 Antibodies(Hybridomas)

Balb/c mice were immunized six times and boosted with CD19-transfectedHEK293 cells (mean receptor density 35,000 per cell). The immuneresponse was monitored by testing serum samples with a CD19-cell-ELISAon human CD19-transfected NIH-3T3 cells. Spleen cells from mice withsufficient titers of anti-human CD19 antibody were used forimmortalization by fusion with mouse myeloma cell line P3X63 Ag8.653.Three fusions were carried out and hybridoma supernatants screened bycell-ELISA on human CD19-transfected NIH-3T3 cells and FACS bindingassay using Daudi (CD19+) and CD19- cells for anti-human CD19 specificantibodies (see Example 1 of WO 2011/147834).

b) Hybridoma Screening and Cell Biological Functional Evaluation ofAnti-CD19 Antibodies

A cell ELISA was applied for screening of hybridomas, and to identifythose hybridomas that secrete antibodies against human-CD19. NIH3T3cells transfected with human-CD19 were used as positive cells;non-transfected NIH3T3 cells were used as negative control cells. Forthe assessment of the positive hybridomas the OD ratio betweentransfected and non-transfected NIH3T3 cells was quantified.

-   -   Culture Medium: DMEM high glucose (4.5 mg/ml), 10% FCS,        Na-Pyruvate, NEAA, Glutamine    -   Antibodies positive control: anti CD19 monoclonal antibody        (IgG1) Pharmingen Cat #555409 c=1 mg/ml    -   Detection antibody: Goat anti-Mouse IgG (H+L) HRP Conjugate        Bio-Rad Cat #170-06516    -   Dilution 1: 2000 in 1× ELISA Blocking Reagent    -   Other reagents: Fibronectin Roche Cat #838039 c=1 mg/ml    -   Glutardialdehyde: 25% stock solution//Grade Agar Scientific        #R102 final concentration: 0.05% in PBS    -   ELISA Blocking Reagent: 10× stock solution//Roche Cat #1112589    -   TMB substrate: Roche Cat #11432559    -   Stop Solution: 1 M H2SO4    -   BioRad Cat #170-6516 Dilution 1:2000 in 1× ELISA Blocking        Reagent

Day 1:

-   -   Fibronectin coating: 5 μg/cm² in PBS; 96 well plate=32 cm²160        μg/plate in 6 ml    -   PBS, 50 μl/well    -   incubate 45 min at RT, aspirate coating solution    -   Seed 1.25×104 cells/well in 50 μl culture medium in a 96 well        plate    -   incubate 40 hours at 37° C.    -   add to upper half of the plate: NIH3T3 cells expressing CD19    -   add to lower half of the plate: non-transfected NIH3T3 cells

Day 3:

-   -   Addition of positive control antibody or samples (supernatant or        mouse serum) in 50 μl culture medium    -   incubate for 2 h at 4° C.    -   Remove medium, fix cells with 100 μl Glutardialdehyde (0.05% in        PBS)    -   Wash two times with 200 μl PBS    -   Addition of detection antibody 1:2000, 50 μl/well    -   incubate 2 h at RT    -   wash three times with 200 μl PBS    -   add 50 μl TMB, incubate for 30 min. at RT,    -   stop by addition of 25 μl M H2SO4; read extinction at 450 nm/620        nm    -   Calculation of results: ratio OD NIH3T3 CD19: OD NIH3T3        non-transfected

The selected antibody demonstrated specific binding to CD19 transfectedNIH3T3 cells as compared to untransfected NIH3T3 cells (see Example 2 ofWO 2011/147834).

c) Humanization of Anti-CD19 Antibody

The CD19 binding specificity of the murine antibody was transferred ontoa human acceptor framework to eliminate potential immunogenicity issuesarising from sequence stretches that the human body will recognize asforeign. This was done by engrafting the entire complementarydetermining regions (CDR) of the murine (donor) antibody onto a human(acceptor) antibody framework, and is called CDR-grafting or antibodyhumanization.

The murine amino acid sequence was aligned with a collection of humangerm-line antibody V genes, and sorted according to sequence identityand homology. Before selecting one particular acceptor sequence, theso-called canonical loop structures of the donor antibody have to bedetermined (Morea, V., et al., Methods, Vol 20, Issue 3 (2000) 267-279).These canonical loop structures are determined by the type of residuespresent at the so-called canonical positions. These positions lie(partially) outside of the CDR regions, and have to be kept functionallyequivalent in the final construct in order to retain the CDRconformation of the parental (donor) antibody. The human germ-linesequence VBASE_VH1_1 was chosen as the acceptor for the heavy chain andsequence VBASE_VK2_5 was chosen for the light chain.

It was found that the wild-type humanized anti-human CD19 antibody 8B8has three deamidation hotspots in the HVR-L1: NSNGNT (SEQ ID NO: 161).Additionally it was found that in the HVR-H2 a further deamidationhotspot is present: KFNG (SEQ ID NO: 162). To address the deamidationhotspot in the HVR-H2 an N (Asn) to Q (Gln) point mutation at position64 (numbering according to Kabat) has been introduced. To address thedeamidation hotspots in the light chain and to obtain a humanizedanti-human CD19 antibody with improved deamidation stability a singlemutation at position 27e from S (serine) to P (proline) (numberingaccording to Kabat) was introduced. Thus, clone 8B8-018 with the CDRs asshown in Table 18 below was generated.

TABLE 11 Comparison of 8B8-018 with humanized wild-type CD19 antibody8B8 variant→ ↓parameter wt 8B8 8B8-018 K_(D) (BIAcore) [nM] 5 6 t_(1/2)[min] — 43.6 human CD19 binding after 46 95 pH 7.4 incubation [%] humanCD19 binding after 90 99 pH 6.0 incubation [%] SEC main peakafter >95 >95 incubation [%]

Additionally, 8B8-018 maintains the cross-reactivity to cynomolgus CD19as shown in the following

TABLE 12 EC50 [μg/ml] wt 8B8 8B8-018 huCD19 ECD 0.087 0.084 cyCD19 ECD0.313 0.255

1.8.2 Preparation, Purification and Characterization of CD19 Antigen FcFusion for Phage Display Campaign

In order to express and purify the human and cynomolgus CD19 ectodomainin a monomeric state (human CD19 see SEQ ID NO:32), the respective DNAfragment was fused to a human IgG1 Fc gene segment containing the “knob”mutations (human: SEQ ID NO: 163; cynomolgus: SEQ ID NO: 165) and wastransfected with an “Fc-hole” (SEQ ID NO: 76) counterpart (Merchant etal., 1998). An IgA cleavage site (PTPPTP) was introduced between theantigen ectodomain and the Fc knob chain. An Avi tag for directedbiotinylation was introduced at the C-terminus of the antigen-Fc knobchain and mutations H435R and Y436F were introduced in the Fc hole forpurification purposes (Jendeberg L. et al, J. Immunological methods,1997). Combination of the antigen-Fc knob chain containing theS354C/T366W mutations (human: SEQ ID NO: 164; cynomolgus: SEQ ID NO:166), with a Fc hole chain containing the Y349C/T366S/L368A/ Y407Vmutations (SEQ ID NO: 80) allows generation of a heterodimeric Fc fusionfragment which includes a single copy of the CD19 ectodomain (in analogyto the 4-1BB construct in FIG. 3C). Table 13 lists the cDNA and aminoacid sequences of the antigen Fc-fusion construct.

TABLE 13 cDNA and Amino acid sequences of monomeric human and cynomolgusCD19 antigen Fc(kih) fusion molecule SEQ ID NO: Antigen Sequence 163Nucleotide GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAG sequence ofCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCC Fc hole chainAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG with HYRFCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAG mutationTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 164 NucleotideCCCGAGGAACCCCTGGTCGTGAAGGTGGAAGAGGGCGACA sequence ofATGCCGTGCTGCAGTGCCTGAAGGGCACCTCCGATGGCCCT human CD19ACCCAGCAGCTGACCTGGTCCAGAGAGAGCCCCCTGAAGC antigen FcCCTTCCTGAAGCTGTCTCTGGGCCTGCCTGGCCTGGGCATC knob chainCATATGAGGCCTCTGGCCATCTGGCTGTTCATCTTCAACGT avi tagGTCCCAGCAGATGGGCGGCTTCTACCTGTGTCAGCCTGGCCCCCCATCTGAGAAGGCTTGGCAGCCTGGCTGGACCGTGAACGTGGAAGGATCCGGCGAGCTGTTCCGGTGGAACGTGTCCGATCTGGGCGGCCTGGGATGCGGCCTGAAGAACAGATCTAGCGAGGGCCCCAGCAGCCCCAGCGGCAAACTGATGAGCCCCAAGCTGTACGTGTGGGCCAAGGACAGACCCGAGATCTGGGAGGGCGAGCCTCCTTGCCTGCCCCCTAGAGACAGCCTGAACCAGAGCCTGAGCCAGGACCTGACAATGGCCCCTGGCAGCACACTGTGGCTGAGCTGTGGCGTGCCACCCGACTCTGTGTCTAGAGGCCCTCTGAGCTGGACCCACGTGCACCCTAAGGGCCCTAAGAGCCTGCTGAGCCTGGAACTGAAGGACGACAGGCCCGCCAGAGATATGTGGGTCATGGAAACCGGCCTGCTGCTGCCTAGAGCCACAGCCCAGGATGCCGGCAAGTACTACTGCCACAGAGGCAACCTGACCATGAGCTTCCACCTGGAAATCACCGCCAGACCCGTGCTGTGGCACTGGCTGCTGAGAACAGGCGGCTGGAAGGTCGACGCTAGCGGTGGTAGTCCGACACCTCCGACACCCGGGGGTGGTTCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGAACGACATCTTCGAGGCCCAGAAGATTGAATGGCACGAG 165 Fc hole chainDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVV with HYRFVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS mutationVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHE ALHNRFTQKSLSLSPGK 166human CD19 PEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPF antigen FcLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSE knob chainKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPS avi tagSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVDASGGSPTPPTPGGGSADKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE 167 NucleotideCCCCAGGAACCCCTGGTCGTGAAGGTGGAAGAGGGCGACA sequence ofATGCCGTGCTCCAGTGCCTGGAAGGCACCTCCGATGGCCCT cynomolgusACACAGCAGCTCGTGTGGTGCAGAGACAGCCCCTTCGAGC CD19 antigenCCTTCCTGAACCTGTCTCTGGGCCTGCCTGGCATGGGCATC Fc knobAGAATGGGCCCTCTGGGCATCTGGCTGCTGATCTTCAACGT chain avi tagGTCCAACCAGACCGGCGGCTTCTACCTGTGTCAGCCTGGCCTGCCAAGCGAGAAGGCTTGGCAGCCTGGATGGACCGTGTCCGTGGAAGGATCTGGCGAGCTGTTCCGGTGGAACGTGTCCGATCTGGGCGGCCTGGGATGCGGCCTGAAGAACAGAAGCAGCGAGGGCCCTAGCAGCCCCAGCGGCAAGCTGAATAGCAGCCAGCTGTACGTGTGGGCCAAGGACAGACCCGAGATGTGGGAGGGCGAGCCTGTGTGTGGCCCCCCTAGAGATAGCCTGAACCAGAGCCTGAGCCAGGACCTGACAATGGCCCCTGGCAGCACACTGTGGCTGAGCTGTGGCGTGCCACCCGACTCTGTGTCCAGAGGCCCTCTGAGCTGGACACACGTGCGGCCAAAGGGCCCTAAGAGCAGCCTGCTGAGCCTGGAACTGAAGGACGACCGGCCCGACCGGGATATGTGGGTGGTGGATACAGGCCTGCTGCTGACCAGAGCCACAGCCCAGGATGCCGGCAAGTACTACTGCCACAGAGGCAACTGGACCAAGAGCTTTTACCTGGAAATCACCGCCAGACCCGCCCTGTGGCACTGGCTGCTGAGAATCGGAGGCTGGAAGGTCGACGCTAGCGGTGGTAGTCCGACACCTCCGACACCCGGGGGTGGTTCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGAACGACATCTTCGAGGCCCAGAAGATTGAATGGCACGAG 168 cynomolgusPQEPLVVKVEEGDNAVLQCLEGTSDGPTQQLVWCRDSPFEPF CD19 antigenLNLSLGLPGMGIRMGPLGIWLLIFNVSNQTGGFYLCQPGLPSE Fc knobKAWQPGWTVSVEGSGELFRWNVSDLGGLGCGLKNRSSEGPS chain avi tagSPSGKLNSSQLYVWAKDRPEMWEGEPVCGPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVRPKGPKSSLLSLELKDDRPDRDMWVVDTGLLLTRATAQDAGKYYCHRGNWTKSFYLEITARPALWHWLLRIGGWKVDASGGSPTPPTPGGGSADKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE

For the production of the monomeric antigen/Fc fusion molecules,exponentially growing suspension CHO cells were co-transfected with twoplasmids encoding the two components of fusion protein (knob and holechains) using standard methods.

Secreted protein was purified from cell culture supernatant by affinitychromatography using Protein A, followed by size exclusionchromatography. For affinity chromatography, the supernatant was loadedon a MabSelect Sure column volume (CV)=5-15 mL, resin from GEHealthcare) equilibrated with Sodium Phosphate (20 mM), Sodium Citrate(20 mM), 0.5M sodium chloride buffer (pH 7.5). Unbound protein wasremoved by washing with at least 6 column volumes of the same buffer.The bound protein was eluted using a linear gradient; step 1, 10 CV from0 to 60% elution buffer (20 mM sodium citrate, 500 mM Sodium chloridebuffer (pH 2.5)); step 2, 2 CV from 60 to 100% elution buffer. For thelinear gradient an additional 2 column volumes step elution with 100%elution buffer was applied.

The pH of collected fractions was adjusted by adding 1/40 (v/v) of 2MTris, pH8.0. The protein was concentrated and filtered prior to loadingon a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 2 mMMOPS, 150 mM sodium chloride, 0.02% (w/v) sodium azide solution of pH7.4.

Table 14 summarizes the yield and final monomer content of monomerichuman and cynomolgus CD19 antigen Fc(kih) fusion protein.

TABLE 14 Biochemical analysis of monomeric human and cynomolgus CD19antigen Fc (kih) fusion protein Monomer [%] Yield Construct (SEC) [mg/l]monomeric human CD19 Fc (kih) fusion protein 91 0.2 monomeric cynomolgusCD19 Fc (kih) fusion 95 3.56 protein

Part of the purified antigen was in vitro biotinylated using the BirAbiotin-protein ligase standard reaction kit (Avidity, Cat. # BirA500)according to the manufacturer's instructions. The biotinylation degreefor the human CD19-containing fusion was 94%, for the respectivecynomolgus CD19 construct 100%. The biotinylated protein was then usedfor selection, screening and characterization of affinity-matured8B8-derived clones devoid of the de-amidation hotspots N27d and N28. Twophage display libraries were generated in which a) both asparagineresidues at positions 27d and 28 were eliminated and b) additional CDRsof heavy and light chain were randomized in order to select for 8B8variants with an improved affinity.

1.8.3 Generation of 8B8 Affinity Maturation Libraries Devoid of CDR-L1Hotspots

Generation of affinity-matured 8B8-derived antibodies without thede-amidation sites N27d and N28, located in CDR-L1, was carried out byphage display using standard protocols (Silacci et al, 2005). In a firststep, the VL and VH DNA sequences of the humanized parental clone 8B8(SEQ ID NO: 169 and SEQ ID NO: 170) were cloned into a phagemid whichwas then used as a template for randomization. In a next step, twolibraries were generated for the selection of favourable clones by phagedisplay. In order to eliminate the above-mentioned hotspot positions, aLCDR1 randomization primer (SEQ ID NO: 171) that only allowed aminoacids S T Q E at positions 27d and 28 was used for both libraries.Maturation library 1 was randomized in CDR1 and 2 of both the light andthe heavy chain, while maturation library 2 was randomized in CDR1 and 3of the light chain and in CDR3 of the heavy chain. For the generation ofthe maturation library 1, randomized in CDR1 and 2 of both the light andthe heavy chain, three fragments were assembled by “splicing byoverlapping extension” (SOE) PCR and cloned into the phage vector. Thefollowing primer combinations were used to generate the libraryfragments: fragment 1 (LMB3 (SEQ ID NO: 176) and CD19 L1 reverse random(SEQ ID NO: 171), fragment 2 (CD19 L2 forward random (SEQ ID NO: 172)and CD19 H1 reverse random (SEQ ID NO: 173), and fragment 3 (CD19 H2forward random (SEQ ID NO: 174) and CD19 H3 reverse constant (SEQ ID NO:175) (Table 15). After assembly of sufficient amounts of full lengthrandomized fragment, it was digested with NcollNhel alongside withidentically treated acceptor phagemid vector. A 3-fold molar excess oflibrary insert was ligated with 10 μg of phagemid vector. Purifiedligations were used for 20 transformations resulting in 2×10 exp9transformants. Phagemid particles displaying the 8B8 affinity maturationlibrary were rescued and purified by PEG/NaCl purification to be usedfor selections.

The generation of the second library, randomized in CDR1 and 3 of thelight chain and in CDR3 of the heavy chain, was done similarly. Thefollowing primer combinations were used to generate the libraryfragments: fragment 1 (LMB3 (SEQ ID NO: 176) and CD19 L1 reverse random(SEQ ID NO: 171), fragment 2 (CD19 L1 forward constant (SEQ ID NO 177)and CD19 L3 reverse random (SEQ ID NO 178), and fragment 3 (CD19 L3forward constant (SEQ ID NO: 179) and CD19 H3 reverse random (SEQ ID NO:180) (Table 16). After assembly of sufficient amounts of full lengthrandomized fragment, it was digested with NcoI/KpnI alongside withidentically treated acceptor phagemid vector. A 3-fold molar excess oflibrary insert was ligated with 20 ug of phagemid vector. Purifiedligations were used for 40 transformations resulting in 2×10 exp9transformants. Phagemid particles displaying the 8B8 affinity maturationlibrary were rescued and purified by PEG/NaCl purification to be usedfor selections.

TABLE 15 Primers for 8B8 affinity maturation and hotspot removal libraryL1_L2/H1_H2 SEQ ID Name Sequence 171 CD19 L1CAG CTG CGG GCT CTG ACC CGG TTT CTG GAG ATA reverseCCA GTT CAG 1 CGT 2 GCC 3 GGA 4 TTC CAG AGA TTG randomGCT GGA TTT GCA AGA AAT G 1: 40% Y, 6% A/S/T/G/P/D/N/E/Q/V, 2: 40% N, 6%A/S/T/Y/G/P/D/E/Q/V, 3: 25% S/T/Q/E, 4: 25% S/T/Q/E 172 CD19 L2CTC CAG AAA CCG GGT CAG AGC CCG CAG CTG CTG forwardATC TAC 5 GTA TCT 6 CGC 7 8 GGC GTT 9 GAT CGT TTC randomAGC GGT TCT GGA TCC GGC ACC5: 30% R, 20% E, 5% A/S/T/Y/G/P/D/N/Q/V. 6: 30% K, 20%S, 5% A/N/T/Y/G/P/D/E/Q/V, 7: 40% F, 5%A/S/T/Y/G/P/D/E/Q/V/I/L, 8: 40% S, 6.6%A/T/Y/G/P/D/E/Q/V, 9: 50% P, 50% L 173 CD19 H1CAT CCA CTC CAG ACC CTG GCC CGG GGC CTG ACG reverseAAC CCA 10 CAT 11 12 13 14 GAA 15 GTA ACC AGA TGC randomTTT GCA GCT CAC TTT AAC GGA AGC10: 52% H, 4% G/A/S/P/T/N/Y/D/E/Q/V/I, 11: 30% I, 15%Y, 5% G/A/S/T/P/N/H/D/E/Q/V, 12: 52% Y, 4%G/A/S/P/T/N/H/D/E/Q/V/I, 13: 30% D, 15% G, 5%A/S/P/Y/N/H/D/E/Q/V/I, 14: 52% T, 4%G/A/S/P/Y/N/H/D/E/Q/V/I, 15: 52% T, 4% G/A/S/P/Y/N/H/D/E/Q/V/I 174CD19 H2 CAG GCC CCG GGC CAG GGT CTG GAG TGG ATG GGC forward16 ATT 17 CCA 18 19 20 21 TCC 22 TAT ACC 23 AAA TTC randomCAG GGC CGC GTC ACG ATG ACC16: 45% Y, 5% A/S/P/T/N/H/D/E/Q/V/I, 17: 52% N, 4%G/A/S/P/Y/T/H/D/E/Q/V/I, 18: 40% Y, 5%G/A/S/P/T/N/H/D/E/Q/V/I, 19: 30% N, 15% S, 5%G/A/T/P/Y/H/D/E/Q/V/I, 20: 30% D, 15% G, 5%A/S/T/P/Y/N/H/E/Q/V/I, 21: 52% G, 4%N/A/S/P/Y/T/H/D/E/Q/V/I, 22: 30% K, 15% N, 4%G/A/S/P/Y/T/H/D/E/Q/V/I, 23: 30% E, 15% Q, 5% G/A/S/T/P/Y/N/H/D/V/I 175CD19 H3 CGTCACCGGTTCGGGGAAGTAGTCCTTGACCAG reverse constant 176 LMB3CAGGAAACAGCTATGACCATGATTAC

TABLE 16 Primers for 8B8 affinity maturation and hotspot removal libraryL1_L3/H3 SEQ ID Name Sequence 177 CD19 L1TGGTATCTCCAGAAACCGGGTCAGAGCCCGCAG forward constant 171 CD19 L1See Table 15 reverse random 178 CD19 L3TTT AAT TTC CAG TTT AGT TCC TTG ACC GAA GGT 24 reverse25 26 27 28 29 CTG CAG ACA ATA GTA GAC GCC AAC random GTC TTC AGC24: 52% Y, 4% G/A/S/T/N/P/D/E/Q/V/L/I, 25: 52% P, 4%G/A/S/T/Y/N/H/D/E/Q/V/I, 26: 42% V, 10% L, 4%G/A/S/T/Y/N/P/D/E/Q/V/I, 27: 52% H, 4%G/A/S/T/Y/N/P/D/E/Q/V/I, 28: 42% T, 10% I, 4%G/A/S/T/Y/N/P/D/E/Q/V/L, 29: 45% L, 11% G, 4% A/S/T/Y/N/P/D/E/Q/V/I 179CD19 L3 ACCTTCGGTCAAGGAACTAAACTGGAAATTAAACG forward constant 180 CD19 H3TT GGT GCT AGC AGA GCT TAC GGT CAC CGT GGT reverseACC TTG GCC CCA GTA ATC AAA 30 31 32 33 34 35 36 37 random38 GCG TGC ACA ATA GTA AAC AGC GGT GTC30: 50% L, 3.8% G/A/S/T/P/H/Y/N/D/E/Q/V/I, 31: 50% A,4.2% G/S/T/P/H/Y/N/D/E/Q/V/I, 32: 50% S, 4.2%G/A/T/P/H/Y/N/D/E/Q/V/I, 33: 50% G, 4.2%S/A/T/P/H/Y/N/D/E/Q/V/I, 34: 50% Y, 4.2%G/A/T/P/H/S/N/D/E/Q/V/I, 35: 50% Y, 4.2%G/A/T/P/H/S/N/D/E/Q/V/I, 36: 50% Y, 4.2%G/A/T/P/H/S/N/D/E/Q/V/I, 37: 50% T, 4.2%G/A/Y/P/H/S/N/D/E/Q/V/I, 38: 50% G, 4.2% Y/A/T/P/H/S/N/D/E/Q/V/I 176LMB3 See Table 15

1.8.4 Selection of Affinity Matured 8B8-derived Clones Devoid of CDR-L1Hotspots N27d and N28

For the selection of affinity-matured clones devoid of theCDR-Llhotspots N27d and N28, two selection approaches by phage displaywere performed:

In the first approach, the selection was executed on human CD19-Fcfusion protein using both phage display libraries. Panning rounds wereperformed in solution according to the following pattern: 1. binding of˜10¹² phagemid particles to 30nM biotinylated CD19-Fc protein for 0.5 hin a total volume of 1 ml, 2. capture of biotinylated CD19-Fc proteinand specifically bound phage particles by addition of 5.4×10⁷streptavidin-coated magnetic beads for 10 min, 3. washing of beads using5×1 ml PBS/Tween20 and 5×1 ml PBS, 4. elution of phage particles byaddition of 1 ml 100 mM TEA for 10 min and neutralization by adding 500ul M Tris/HCl pH 7.4, 5. re-infection of exponentially growing E. coliTG1 bacteria, and 6.infection with helperphage VCSM13 and subsequentPEG/NaCl precipitation of phagemid particles to be used in subsequentselection rounds. Selections were carried out over 3 rounds usingdecreasing antigen concentrations (30×10⁻⁹M, 10×10⁻⁹M, and 3×10⁻⁹M). Inround 2 and 3, capture of antigen:phage complexes was performed usingneutravidin plates instead of streptavidin beads. Neutravidin plateswere washed with 5× PBS/Tween20 and 5× PBS. In round 3, the neutravidinplate was incubated overnight in 2 liters PBS for an “off-rate”selection before phage was eluted from the plate. Furthermore,cynomolgus CD19-Fc protein was used in round 2 in order to enrichcross-reactive binders.

In the second selection approach, the phage panning was executed oncells transiently expressing either the human or cynomolgus CD19 ECD onthe cell surface. For the transient transfection of HEK cells,expression plasmids were generated that harbor the DNA sequences (from5′ to 3′) for the following protein segments: A Flag tag, a SNAP tag,the CD19 ECD of either human or cynomolgus origin, and the transmembraneregion of the Platelet-derived growth factor receptor (PDGFR) (SEQ IDNOs: 227 and 228). The expression of the respective proteins (SEQ IDNOs: 229 and 230) on the cell surface was confirmed by flow cytometryusing an anti-Flag antibody for detection. Both libraries were exposedin the first selection round to cells either expressing the human orcynomolgus CD19 ECD-containing protein fusion. For the subsequentpanning rounds, the species of the CD19 ECD was alternated accordingly.Cells transiently transfected with an irrelevant membrane protein wereused for pre-clearing.

Panning rounds were performed according to the following pattern:

-   1. Transfection of HEK cells with constructs expressing either CD19    ECD or an irrelevant transmembrane protein according to the standard    procedure described before,-   2. Incubation of the cells for total 48h at 37° C. in an incubator    with a 5% CO₂ atmosphere,-   3. Isolation of cells by centrifugation (3 min at 250×g) and    re-suspension of 1×10E7 CD19 ECD-positive cells and 1×10E7 negative    cells in PBS/5% BSA, respectively,-   4. Pre-clearing of unspecific phage by incubating the phage library    with 1×107 CD19-negative cells for 60 min at 4° C. using a gently    rotating tube rotator,-   5. Centrifugation of cells at 250xg for 3min and transfer of    supernatant into a fresh tube and addition of 1×10E7 CD19-positive    cells and incubation for 60 min at 4° C. by gentle rotation on a    tube rotator,-   6. Washing of cells by centrifugation for 1 min at 250×g, aspiration    of the supernatant, and re-suspension in 1 ml PBS (8 times),-   7. Phage elution with 1 ml 100mM TEA, incubation for 5 min at RT,    and neutralization of the eluate with 500 ul 1M Tris-HCl, pH7.6,-   8. re-infection of exponentially growing E. coli TG1 bacteria, and-   9. infection with helperphage VCSM13 and subsequent PEG/NaCl    precipitation of phagemid particles to be used in subsequent    selection rounds. Selections were carried out over 3 rounds.

For both selection approaches, specific binders were identified by ELISAas follows: 100 ul of 30 nM biotinylated CD19-Fc protein per well werecoated on neutravidin plates. Fab-containing bacterial supernatants wereadded and binding Fabs were detected via their Flag-tags using ananti-Flag/HRP secondary antibody.

Clones that were ELISA-positive on recombinant human CD19 were furthertested in a cell-based ELISA using cells that were transientlytransfected with the human CD19 ECD-containing expression plasmid (SEQID NO: 227). This analysis was performed as follows: 48 h aftertransfection, HEK cells were harvested and centrifuged at 250×g for 5min. Cells were then re suspended in ice-cold PBS BSA 2% to 4×10⁶cells/ml and incubated for 20 min on ice to block unspecific bindingsites. 4×10⁵ cells in 100 ul were distributed to each well of a 96 wellplate and centrifuged at 250×g and 4° C. for 3 min. Supernatant wasaspirated off and 50 ul bacterial supernatant containing soluble Fabfragments was diluted with 50 ul ice-cold PBS/BSA 2%, added to theplate, mixed with the cells and incubated for 1 h at 4° C. Afterwards,cells were washed 3 times with ice cold PBS before 100 ul PBS BSA 2% perwell containing a 1:2000 dilution of anti-Fab-HRP antibody were added.After an incubation time of 1 h, cells were washed again 3 times withice-cold PBS. For the development, 100 ul “1-step ultra TMB-ELISA”substrate was added per well. After an incubation time of 10 minutes,supernatant was transferred to a new 96-well plate containing 40 ulH₂SO4 1M per well and absorbance was measured 450 nM. Clones exhibitingsignificant signals over background were subjected to a kineticscreening experiment by SPR-analysis using ProteOn XPR36.

1.8.5 Identification of Affinity-Matured 8B8-derived Variants by SPR

In order to further characterize the ELISA-positive clones, the off-ratewas measured by surface plasmon resonance and compared with the parentalhumanized clone 8B8.

For this experiment, 7000 RU of polyclonal anti-human Fab antibody wereimmobilized on all 6 channels of a GLM chip by Amine coupling (NaAcetatepH4.5, 25 μl/min, 240 s) (vertical orientation). Eachantibody-containing bacterial supernatant was filtered and 2-folddiluted with PBS, and then injected for 360s at 25 μl/minute to achieveimmobilization levels of between 100 and 400 response units (RU) invertical orientation. Injection of monomeric CD19-Fc: For one-shotkinetics measurements, injection direction was changed to horizontalorientation, three-fold dilution series of purified monomeric CD19-Fc(varying concentration ranges between 150 and 6 nM) were injectedsimultaneously at 50 μl/min along separate channels 1-4, withassociation times of 180 s, and dissociation times of 300 s. A human IgGFc fragment (150nM) was injected in channel 5 as a negative control forspecific binding to the affinity matured CD19 variants along with a PBSinjection in the 6th channel to provide an “in-line” blank forreferencing. Regeneration was performed by two pulses of 10 mM glycinepH 1.5 and 50 mM NaOH for 30 s at 90 ul/min (horizontal orientation).Dissociation rate constants (k_(off)) were calculated using a simpleone-to-one Langmuir binding model in ProteOn Manager v3.1 software bysimultaneously fitting the sensorgrams. Clones expressing Fabs with theslowest dissociation rate constants were identified. The variabledomains of the corresponding phagemids were sequenced. Importantly, bothasparagine residue in CDR-L1 (position 27d and 28) were replaced by aserine or a threonine, demonstrating that both de-amidation sites wereremoved.

TABLE 17 Dissociation constants of parental 8B8 and selected clone 2B11obtained in screening analysis with bacterial supernatant Dissociationclone constant kd (1/s) Parental 8B8 3.01E−4 2B11 4.37E−6

Table 18 shows the amino acid sequences of the CDRs and variable regionsVH and VL of clones 8B8-018 and 8B8-2B11, respectively.

TABLE 18 Sequences of of the CDRs and variable regionsVH and VL of clones 8B8-018 and 8B8-2B11 SEQ ID NO: Description Sequence120 CD19 (8B8-018) CDR-H1 DYIMH 121 CD19 (8B8-018) CDR-H2YINPYNDGSKYTEKFQG 122 CD19 (8B8-018) CDR-H3 GTYYYGSALFDY 123CD19 (8B8-018) CDR-L1 KSSQSLENPNGNTYLN 124 CD19 (8B8-018) CDR-L2 RVSKRFS125 CD19 (8B8-018) CDR-L3 LQLTHVPYT 126 CD19 (8B8-018) VHQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMH WVRQAPGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGS ALFDYWGQGTTVTVSS 127CD19 (8B8-018) VL DIVMTQTPLSLSVTPGQPASISCKSSQSLENPNGNTYLNWYLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQLTHVPYTFGQGTKLEIK 128 CD19 (8B8-2B11) CDR-H1 DYIMH129 CD19 (8B8-2B11) CDR-H2 YINPYNDGSKYTEKFQG 130 CD19 (8B8-2B11) CDR-H3GTYYYGPQLFDY 131 CD19 (8B8-2B11) CDR-L1 KS SQSLETSTGTTYLN 132CD19 (8B8-2B11) CDR-L2 RVSKRFS 133 CD19 (8B8-2B11) CDR-L3 LQLLEDPYT 134CD19 (8B8-018) VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQAPGQGLEWMGYINPYNDGSKYTEKFQGRVT MTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGPQLFDYWGQGTTVTVSS 135 CD19 (8B8-018) VLDIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGTTYLNWYLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQLLEDPYTFGQGTKLEIK

Example 2 Preparation and Purification of “Untargeted” Single Chain4-1BB Ligand Trimer-Containing Fc Fusion Antigen Binding Molecules

The polypeptide chain coding for the single chain trimeric ligand (FIG.1A) was used to prepare untargeted (germline control, DP47) 4-1BB ligandtrimer-containing Fc fusion antigen binding molecules (for example themonovalent control shown in FIG. 3B), as described above for theFAP-targeted construct.

The variable region of heavy and light chain DNA sequences of the FAPbinder, were replaced with those of a germline control (DP47) andsubcloned in frame with either the constant heavy chain of the hole orthe constant light chain of human IgG1.

The untargeted single chain 4-1BB ligand trimer-containing Fc (kih)fusion antigen binding molecules were produced as described above forthe FAP-targeted construct. The cells were transfected with thecorresponding expression vectors at a 1:1:1 ratio (“vector trimericligand-knob chain”: “vector DP47 Fab-hole chain”: “vector DP47 lightchain”).

Table 19 shows, respectively, the cDNA and amino acid sequences of themonovalent DP47-containing 4-1BB ligand trimer-containing Fc (kih)fusion antigen binding molecule.

TABLE 19Sequences of monovalent DP47-containing single chain 4-1BB ligandtrimer-containing Fc (kih) fusion antigen binding molecule (DP47single chain 4-1BBL trimer) (Control 1) SEQ ID NO: Description Sequence66 nucleotide see Table 1 sequence of trimeric hu 4- 1BBL (71-254)-Fc knob chain 69 nucleotide GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACsequence of DP47 AGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCC Fc hole chainGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGCAGCGGATTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCC TCTCCCTGTCTCCGGGTAAA 70 nucleotideGAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTT sequence of DP47GTCTCCAGGGGAAAGAGCCACCCTCTCTTGCAGGGCCA light chainGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGCTGACGTTCGGCCAGGGGACCAAAGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAG AGTGT 15 trimeric hu 4-see Table 1 1BBL (71-254)- Fc knob chain 71 DP47 Fc holeEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQ chainAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK 72DP47 light chain EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC

Table 20 summarizes the yield and final monomer content of theDP47-containing single chain 4-1BB ligand trimer-containing Fc (kih)fusion molecule.

TABLE 20 Summary of the production of DP47-containing 4-1BBLtrimer-containing Fc (kih) fusion molecule Monomer [%] Yield LC/MSConstruct (SEC) [mg/l] (non red) DP47-containing 4-1BBL 100 8Theoretical*: trimer-containing Fc 156912.9 Da (kih) fusion moleculeExperimental: (DP47 single chain 156937.5 Da 4-1BBL trimer) *withoutControl 1 terminal lysines

Table 21 shows, respectively, the cDNA and amino acid sequences of thebivalent DP47-containing 4-1BB ligand trimer-containing Fc (kih) fusionantigen binding molecule (Control 2).

TABLE 21 Sequences of bivalent DP47-containing single chain 4-1BB ligandtrimer-containing Fc (kih) fusion antigen binding molecule (DP47single chain 4-1BBL trimer) (Control 2) SEQ ID NO: Description Sequence 69 nucleotide see Table 19 sequence of DP47 Fc hole chain 181nucleotide GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTAC sequence of DP47AGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCC Fc knob fused toGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCG trimeric hu 4-CCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCT 1BBL (71-254)ATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGCAGCGGATTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGCCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACTCCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCTGGCGGAGGCGGAGGATCTGGCGGGGGAGGATCTAGAGAGGGCCCTGAGCTGTCCCCCGATGATCCTGCTGGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGCTGATCGACGGCCCTCTGAGCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAGGGATCTGGATCTGTGTCTCTGGCCCTGCATCTGCAGCCCCTGAGATCAGCTGCTGGCGCTGCTGCTCTGGCTCTGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAAGCCAGAGCCAGGCACGCTTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCTCCAAGAAGCGAAGGCGGCGGAGGCTCCGGAGGAGGCGGCTCTAGAGAAGGACCTGAACTGAGCCCAGACGACCCCGCAGGGCTGCTGGATCTGAGACAGGGAATGTTCGCCCAGCTGGTGGCTCAGAATGTGCTGCTGATTGATGGACCCCTGTCCTGGTACTCCGACCCAGGGCTGGCAGGGGTGTCCCTGACTGGCGGACTGTCTTACAAAGAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAAGGCAGTGGCTCTGTGTCCCTGGCACTGCATCTGCAGCCTCTGCGCAGTGCTGCAGGCGCAGCCGCACTGGCACTGACTGTGGACCTGCCCCCAGCTTCCAGCGAGGCTAGAAACAGCGCCTTCGGGTTTCAAGGACGCCTGCTGCATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCATACTGAGGCTAGGGCCAGACATGCCTGGCAGCTGACCCAGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACACCTGAGATCCCCGCTGGCCTGCCAAGCCCTAGATCAGAAGGCGGAGGGGGATCAGGGGGAGGCGGATCCAGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCTCCAAGAAGCGAA  70 nucleotide see Table 19sequence of DP47 light chain  71 DP47 Fc hole see Table 19 chain 182DP47 Fc knob EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQ fused to trimericAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTL hu 4-1BBL (71-YLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSAS 254)TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLEILSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVT PEIPAGLPSPRSE  72DP47 light chain see Table 19

Example 3 Production of Targeted Single Chain 4-1BB LigandTrimer-Containing Fc (kih) Fusion Antigen Binding Molecules and TheirControls

The encoding sequences of the targeted and untargeted trimeric 4-1BBligand Fc (kih) fusion antigen binding molecules as described hereinbefore were cloned into a plasmid vector, which drives expression of theinsert from an MPSV promoter and contains a synthetic polyA sequencelocated at the 3′ end of the CDS. In addition, the vector contains anEBV OriP sequence for episomal maintenance of the plasmid.

The targeted and untargeted single chain trimeric 4-1BB ligand Fc (kih)fusion antigen binding molecules were produced by co-transfectingHEK293-EBNA cells with the mammalian expression vectors usingpolyethylenimine. The cells were transfected with the correspondingexpression vectors in a 1:1:1 ratio (“vector knob chain”: “vector holechain”: “vector light chain”).

For production in 500 mL shake flasks, 300 million HEK293 EBNA cellswere seeded 24 hours before transfection. For transfection cells werecentrifuged for 10 minutes at 210×g, and the supernatant was replaced by20mL pre-warmed CD CHO medium. Expression vectors (200 μg of total DNA)were mixed in 20 mL CD CHO medium. After addition of 540 μL PEI, thesolution was vortexed for 15 seconds and incubated for 10 minutes atroom temperature. Afterwards, cells were mixed with the DNA/PEIsolution, transferred to a 500 mL shake flask and incubated for 3 hoursat 37° C. in an incubator with a 5% CO₂ atmosphere. After theincubation, 160 mL of Excell medium supplemented with 6mM L-Glutamine, 5g/L PEPSOY and 1.2 mM valproic acid was added and cells were culturedfor 24 hours. One day after transfection 12% Feed were added. Afterculturing for 7 days, the supernatant was collected by centrifugationfor 30-40 minutes at least 400×g. The solution was sterile filtered(0.22 μm filter), supplemented with sodium azide to a finalconcentration of 0.01% (w/v), and kept at 4° C.

Secreted proteins were purified from cell culture supernatants byaffinity chromatography using Protein A, followed by size exclusionchromatography. For affinity chromatography, the supernatant was loadedon a MabSelect Sure column (CV=5-15 mL, resin from GE Healthcare)equilibrated with Sodium Phosphate (20 mM), Sodium Citrate (20 mM)buffer (pH 7.5). Unbound protein was removed by washing with at least 6column volumes of the same buffer. The bound protein was eluted usingeither a linear gradient (20 CV) or a step elution (8 CV) with 20 mMsodium citrate, 100 mM Sodium chloride, 100 mM Glycine buffer (pH 3.0).For the linear gradient an additional 4 column volumes step elution wasapplied.

The pH of collected fractions was adjusted by adding 1/10 (v/v) of 0.5Msodium phosphate, pH8.0. The protein was concentrated prior to loadingon a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 20 mMhistidine, 140 mM sodium chloride, 0.01% (v/v) Tween20 solution of pH6.0.

The protein concentration was determined by measuring the opticaldensity (OD) at 280 nm, using a molar extinction coefficient calculatedon the basis of the amino acid sequence.

Purity and molecular weight of the targeted trimeric 4-1BB ligand Fc(kih) fusion was analyzed by SDS-PAGE in the presence and absence of areducing agent (5 mM 1,4-dithiotreitol) and staining with CoomassieSimpleBlue™ SafeStain (Invitrogen USA) or CE-SDS using Caliper LabChipGXII (Perkin Elmer). The aggregate content of samples was analyzed usinga TSKgel G3000 SW XL analytical size-exclusion column (Tosoh)equilibrated in 25 mM K₂HPO₄, 125 mM NaCl, 200 mM L-ArginineMonohydrocloride, 0.02% (w/v) NaN₃, pH 6.7 running buffer at 25° C.

Table 22 summarizes the yield and final monomer content of the targetedand untargeted single chain trimeric 4-1BB ligand Fc (kih) fusionantigen binding molecules.

TABLE 22 Biochemical analysis of targeted and untargeted single chain4-1BB ligand trimer-containing Fc (kih) fusion antigen binding moleculesMonomer [%] Yield Construct (SEC) [mg/l] Bivalent FAP(4B9) targetedsingle chain 92.2 2.7 trimeric 4-1BB ligand Fc (kih) fusion antigenbinding molecule Compound S2 Bivalent CEA(sm9b) targeted single chain98.9 3.9 trimeric 4-1BB ligand Fc (kih) fusion antigen binding moleculeCompound S4 Bivalent untargeted trimeric 4-1BB single 89 2.0 chainligand Fc fusion as control antigen binding molecule Control 2Monovalent FAP(4B9) targeted single chain 97.5 21.2 trimeric 4-1BBligand Fc (kih) fusion antigen binding molecule Compound S3 MonovalentCEA(sm9b) targeted single chain 94.5 4.7 trimeric 4-1BB ligand Fc (kih)fusion antigen binding molecule Compound S5

Example 4 Preparation, Purification and Characterization of 4-1BB

DNA sequences encoding the ectodomains of human, mouse or cynomolgus4-1BB (Table 23) were subcloned in frame with the human IgG1 heavy chainCH2 and CH3 domains on the knob (Merchant et al., 1998). An AcTEVprotease cleavage site was introduced between an antigen ectodomain andthe Fc of human IgG1. An Avi tag for directed biotinylation wasintroduced at the C-terminus of the antigen-Fc knob. Combination of theantigen-Fc knob chain containing the S354C/T366W mutations, with a Fchole chain containing the Y349C/T366S/L368A/Y407V mutations allowsgeneration of a heterodimer which includes a single copy of the 4-1BBectodomain containing chain, thus creating a monomeric form of Fc-linkedantigen (FIG. 3C). Table 24 lists the cDNA and amino acid sequences ofmonomeric antigen Fc(kih) fusion molecules as depicted in FIG. 3C.

TABLE 23 Amino acid numbering of antigen ectodomains (ECD) and theirorigin SEQ ID NO: Construct Origin ECD 73 human 4-1BB ECD Synthetizedaccording aa 24-186 to Q07011 74 cynomolgus 4-1BB isolated from aa24-186 ECD cynomolgus blood 75 murine 4-1BB Synthetized according aa24-187 ECD to P20334

TABLE 24 cDNA and Amino acid sequences of monomeric antigen Fc(kih)fusion molecules (produced by combination of one Fc hole chainwith one antigen Fc knob chain) SEQ ID NO: Antigen Sequence 76Nucleotide GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGA sequenceACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAA Fc hole chainACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGC AGAAGAGCCTCTCCCTGTCTCCGGGTAAA 77Nucleotide CTGCAGGACCCCTGCAGCAACTGCCCTGCCGGCACCTTC sequenceTGCGACAACAACCGGAACCAGATCTGCAGCCCCTGCCC human 4-1BBCCCCAACAGCTTCAGCTCTGCCGGCGGACAGCGGACCT antigen Fc knobGCGACATCTGCAGACAGTGCAAGGGCGTGTTCAGAACC chainCGGAAAGAGTGCAGCAGCACCAGCAACGCCGAGTGCGACTGCACCCCCGGCTTCCATTGTCTGGGAGCCGGCTGCAGCATGTGCGAGCAGGACTGCAAGCAGGGCCAGGAACTGACCAAGAAGGGCTGCAAGGACTGCTGCTTCGGCACCTTCAACGACCAGAAGCGGGGCATCTGCCGGCCCTGGACCAACTGTAGCCTGGACGGCAAGAGCGTGCTGGTCAACGGCACCAAAGAACGGGACGTCGTGTGCGGCCCCAGCCCTGCTGATCTGTCTCCTGGGGCCAGCAGCGTGACCCCTCCTGCCCCTGCCAGAGAGCCTGGCCACTCTCCTCAGGTCGACGAACAGTTATATTTTCAGGGCGGCTCACCCAAATCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGAACGACATCTTCGAGGCCCAGAAGATTGAATGGCACGAG 78 NucleotideTTGCAGGATCTGTGTAGTAACTGCCCAGCTGGTACATTC sequenceTGTGATAATAACAGGAGTCAGATTTGCAGTCCCTGTCCT cynomolgus 4-CCAAATAGTTTCTCCAGCGCAGGTGGACAAAGGACCTGT 1BB antigenGACATATGCAGGCAGTGTAAAGGTGTTTTCAAGACCAG Fc knob chainGAAGGAGTGTTCCTCCACCAGCAATGCAGAGTGTGACTGCATTTCAGGGTATCACTGCCTGGGGGCAGAGTGCAGCATGTGTGAACAGGATTGTAAACAAGGTCAAGAATTGACAAAAAAAGGTTGTAAAGACTGTTGCTTTGGGACATTTAATGACCAGAAACGTGGCATCTGTCGCCCCTGGACAAACTGTTCTTTGGATGGAAAGTCTGTGCTTGTGAATGGGACGAAGGAGAGGGACGTGGTCTGCGGACCATCTCCAGCCGACCTCTCTCCAGGAGCATCCTCTGCGACCCCGCCTGCCCCTGCGAGAGAGCCAGGACACTCTCCGCAGGTCGACGAACAGTTATATTTTCAGGGCGGCTCACCCAAATCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGAACGACATCTTCGAGGCCCAGAAGATTGAATGGCACGAG 79 murine 4-1BBGTGCAGAACAGCTGCGACAACTGCCAGCCCGGCACCTT antigen Fc knobCTGCCGGAAGTACAACCCCGTGTGCAAGAGCTGCCCCC chainCCAGCACCTTCAGCAGCATCGGCGGCCAGCCCAACTGCAACATCTGCAGAGTGTGCGCCGGCTACTTCCGGTTCAAGAAGTTCTGCAGCAGCACCCACAACGCCGAGTGCGAGTGCATCGAGGGCTTCCACTGCCTGGGCCCCCAGTGCACCAGATGCGAGAAGGACTGCAGACCCGGCCAGGAACTGACCAAGCAGGGCTGTAAGACCTGCAGCCTGGGCACCTTCAACGACCAGAACGGGACCGGCGTGTGCCGGCCTTGGACCAATTGCAGCCTGGACGGGAGAAGCGTGCTGAAAACCGGCACCACCGAGAAGGACGTCGTGTGCGGCCCTCCCGTGGTGTCCTTCAGCCCTAGCACCACCATCAGCGTGACCCCTGAAGGCGGCCCTGGCGGACACTCTCTGCAGGTCCTGGTCGACGAACAGTTATATTTTCAGGGCGGCTCACCCAAATCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGAACGACATCTTCGAGGCCCAGAAGATTGAATGGCACG AG 80 Fc hole chainDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK 81human 4-1BB LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDI antigen Fc knobCRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQ chainDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKSGGLNDIFEAQKIEWHE 82cynomolgus 4- LQDLCSNCPAGTFCDNNRSQICSPCPPNSFSSAGGQRTCDIC 1BB antigenRQCKGVFKTRKECSSTSNAECDCISGYHCLGAECSMCEQD Fc knob chainCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSATPPAPAREPGHSPQVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKSGGLNDIFEAQKIEWHE 83murine 4-1BB VQNSCDNCQPGTFCRKYNPVCKSCPPSTFSSIGGQPNCNIC antigen Fc knobRVCAGYFRFKKFCSSTHNAECECIEGFHCLGPQCTRCEKDC chainRPGQELTKQGCKTCSLGTFNDQNGTGVCRPWTNCSLDGRSVLKTGTTEKDVVCGPPVVSFSPSTTISVTPEGGPGGHSLQVLVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKSGGLNDIFEAQKIEWHE

All 4-1BB-Fc-fusion encoding sequences were cloned into a plasmidvector, which drives expression of the insert from an MPSV promoter andcontains a synthetic polyA signal sequence located at the 3′ end of theCDS. In addition, the vector contains an EBV OriP sequence for episomalmaintenance of the plasmid.

For preparation of the biotinylated monomeric antigen/Fc fusionmolecules, exponentially growing suspension HEK293 EBNA cells wereco-transfected with three vectors encoding the two components of fusionprotein (knob and hole chains) as well as BirA, an enzyme necessary forthe biotinylation reaction. The corresponding vectors were used at a2:1:0.05 ratio (“antigen ECD-AcTEV-Fc knob” : “Fc hole”:“BirA”).

For protein production in 500 ml shake flasks, 400 million HEK293 EBNAcells were seeded 24 hours before transfection. For transfection cellswere centrifuged for 5 minutes at 210 g, and supernatant was replaced bypre-warmed CD CHO medium. Expression vectors were resuspended in 20 mLof CD CHO medium containing 200 μg of vector DNA. After addition of 540μL of polyethylenimine (PEI), the solution was vortexed for 15 secondsand incubated for 10 minutes at room temperature. Afterwards, cells weremixed with the DNA/PEI solution, transferred to a 500 mL shake flask andincubated for 3 hours at 37° C. in an incubator with a 5% CO₂atmosphere. After the incubation, 160 mL of F17 medium was added andcells were cultured for 24 hours. One day after transfection, 1 mMvalproic acid and 7% Feed were added to the culture. After 7 days ofculturing, the cell supernatant was collected by spinning down cells for15 min at 210 g. The solution was sterile filtered (0.22 μm filter),supplemented with sodium azide to a final concentration of 0.01% (w/v),and kept at 4° C.

Secreted proteins were purified from cell culture supernatants byaffinity chromatography using Protein A, followed by size exclusionchromatography. For affinity chromatography, the supernatant was loadedon a HiTrap ProteinA HP column (CV=5 mL, GE Healthcare) equilibratedwith 40 mL 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5. Unboundprotein was removed by washing with at least 10 column volumes of 20 mMsodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride containingbuffer (pH 7.5). The bound protein was eluted using a linear pH-gradientof sodium chloride (from 0 to 500 mM) created over 20 column volumesof20 mM sodium citrate, 0.01% (v/v) Tween-20, pH 3.0 . The column wasthen washed with 10 column volumes of 20 mM sodium citrate, 500 mMsodium chloride, 0.01% (v/v) Tween-20, pH 3.0.

The pH of collected fractions was adjusted by adding 1/40 (v/v) of 2MTris, pH8.0. The protein was concentrated and filtered prior to loadingon a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 2 mMMOPS, 150 mM sodium chloride, 0.02% (w/v) sodium azide solution of pH7.4.

For affinity determination to the human receptor, the ectodomain ofhuman 4-1BB was also subcloned in frame with an avi (GLNDIFEAQKIEWHE,SEQ ID NO:84) and a hexahistidine tag. Protein production was performedas described above for the Fc-fusion protein.

Secreted proteins were purified from cell culture supernatants bychelating chromatography, followed by size exclusion chromatography. Thefirst chromatographic step was performed on a NiNTA Superflow Cartridge(5 ml, Qiagen) equilibrated in 20 mM sodium phosphate, 500 nM sodiumchloride, pH 7.4. Elution was performed by applying a gradient over 12column volume from 5% to 45% of elution buffer (20 mM sodium phosphate,500 nM sodium chloride, 500 mM Imidazole, pH 7.4). The protein wasconcentrated and filtered prior to loading on a HiLoad Superdex 75column (GE Healthcare) equilibrated with 2 mM MOPS, 150 mM sodiumchloride, 0.02% (w/v) sodium azide solution of pH 7.4.

TABLE 25 Sequences of monomeric human 4-1BB His molecule SEQ ID NO:antigen Sequence 85 nucleotide CTGCAGGACCCCTGCAGCAACTGCCCTGCCGGCACCTTCTGsequence CGACAACAACCGGAACCAGATCTGCAGCCCCTGCCCCCCC humanAACAGCTTCAGCTCTGCCGGCGGACAGCGGACCTGCGACA 4-1BB HisTCTGCAGACAGTGCAAGGGCGTGTTCAGAACCCGGAAAGAGTGCAGCAGCACCAGCAACGCCGAGTGCGACTGCACCCCCGGCTTCCATTGTCTGGGAGCCGGCTGCAGCATGTGCGAGCAGGACTGCAAGCAGGGCCAGGAACTGACCAAGAAGGGCTGCAAGGACTGCTGCTTCGGCACCTTCAACGACCAGAAGCGGGGCATCTGCCGGCCCTGGACCAACTGTAGCCTGGACGGCAAGAGCGTGCTGGTCAACGGCACCAAAGAACGGGACGTCGTGTGCGGCCCCAGCCCTGCTGATCTGTCTCCTGGGGCCAGCAGCGTGACCCCTCCTGCCCCTGCCAGAGAGCCTGGCCACTCTCCTCAGGTCGACGAACAGTTATATTTTCAGGGCGGCTCAGGCCTGAACGACATCTTCGAGGCCCAGAAGATCGAGTGGCA CGAGGCTCGAGCTCACCACCATCACCATCAC86 human LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICR 4-1BB HisQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQCQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQVDEQLYFQGGSGLNDIFEAQKIEWHEARAHHHHHH

Example 5 Biochemical Characterization of FAP-targeted Single Chain4-1BB Ligand Trimer-Containing Fc Fusion Antigen Binding Molecule bySurface Plasmon Resonance

Binding of FAP-targeted single chain 4-1BB ligand trimer-containing Fc(kih) fusion antigen binding molecule to recombinant 4-1BB was assessedby surface plasmon resonance (SPR). All SPR experiments were performedon a Biacore T100 at 25° C. with HBS-EP as a running buffer (0.01 MHEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore,Freiburg/Germany).

The avidity of the interaction between the FAP-targeted or “untargeted”4-1BB ligand trimer-containing Fc (kih) fusion antigen binding moleculesand recombinant 4-1BB (human, cyno and murine) was determined asillustrated in FIG. 5A. The data demonstrated that all variants of 4-1BBligand trimer-containing Fc (kih) fusion antigen binding molecules (seefor example the curves for FAP (28H1) single chain 4-1BBL trimer(Compound S1) in FIGS. 5A-D, and for the “untargeted” DP47 single chain4-1BBL trimer (Control A) in FIGS. 6A-C) bind with comparable aviditiesto human and cynomolgus 4-1BB but negligibly to the mouse homolog.

Recombinant biotinylated human, cynomolgus and murine 4-1BB Fc(kih)fusion molecules were directly coupled on a SA chip using the standardcoupling instruction (Biacore, Freiburg/Germany). The immobilizationlevel was about 30 resonance units (RU). FAP-targeted 4-1BB ligandtrimer-containing Fc (kih) fusion antigen binding molecule (CompoundS1), or the DP47-containing control, was passed at a concentration rangefrom 0.9 to 500 nM with a flow of 30 μL/minutes through the flow cellsover 180 seconds. The dissociation was monitored for 180 seconds. Bulkrefractive index differences were corrected for by subtracting theresponse obtained on a reference empty flow cell.

For affinity measurement (FIGS. 7A-E), direct coupling of around 7200resonance units (RU) of the anti-human Fc specific antibody wasperformed on a CMS chip at pH 5.0 using the standard amine coupling kit(GE Healthcare). FAP-targeted or untargeted single chain 4-1BB ligandtrimer-containing Fc (kih) fusion antigen binding molecule at 50 nM wascaptured with a flow rate of 30 μl/min for 60 seconds on flow cell 2. Adilution series (1.95 to 1000 nM) of hu4-1BB avi His was passed on bothflow cells at 30 μl/min for 120 seconds to record the association phase.The dissociation phase was monitored for 180 seconds and triggered byswitching from the sample solution to HBS-EP. The chip surface wasregenerated after every cycle using a double injection of 60 seconds 3MMgCl₂. Bulk refractive index differences were corrected for bysubtracting the response obtained on the reference flow cell 1. For theinteraction between targeted single chain trimeric 4-1BB ligand Fc(kih)fusion antigen binding molecule and hu4-1BB avi His, the affinityconstants were derived from the rate constants by fitting to a 1:1Langmuir binding using the Biaeval software (GE Healthcare).Dissociation constants can be found in Table 26.

TABLE 26 Fittings to 1:1 Langmuir binding and Affinity constants LigandAnalyte ka (1/Ms) kd (1/s) KD (M) FAP single chain Hu 4-1BB 6.2E+043.3E−02 5.4E−07 4-1BBL trimer avi His Compound S1 DP47 single chain Hu4-1BB 6.2E+04 3.3E−02 5.2E−07 4-1BBL trimer avi His Control 1

Example 6 Preparation and Purification Of Targeted Single Chain OX40Ligand Trimer-Containing Fc Fusion Antigen Binding Molecules

The DNA sequence encoding part of the ectodomain (amino acids 51-183) ofhuman OX40 ligand was synthetized according to UniProt Accession No.P23510 (SEQ ID NO:38). Two Asn-linked glycosylation sites (N90 and N114)were replaced by aspartic acid (Asp) by mutagenesis.

A polypeptide containing three ectodomains of OX40 ligand, separated by(G45)₂ linkers, was cloned as described in FIG. 1B: human OX40 ligand,(G45)₂ connector, human Ox40 ligand, (G45)₂ connector, human Ox40ligand.

The polypeptide encoding the single chain trimeric Ox40 ligand wassubcloned in frame with the human IgG1 heavy chain CH2 and CH3 domainson the knob (Merchant, Zhu et al. 1998), using a linker (GSPGSSSSGS) ofSEQ ID NO:57.

The variable region of heavy and light chain DNA sequences encoding abinder specific for fibroblast activation protein (FAP) were subclonedin frame with either the constant heavy chain of the hole (Carter, 2001)or the constant light chain of human IgG1.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have been introduced inthe constant region of the knob and hole heavy chains to abrogatebinding to Fc gamma receptors according to the method described inInternational Patent Appl. Publ. No. WO 2012/130831 A1.

Combination of the single chain ligand-Fc knob chain containing theS354C/T366W mutations, with the targeted anti-FAP-Fc hole chaincontaining the Y349C/T366S/L368A/Y407V mutations and the anti-FAP lightchain, allows generation of a heterodimer, which includes a single chaintrimeric OX40 ligand and a FAP binding Fab (FIG. 4A).

Table 27 shows, respectively, the cDNA and amino acid sequences of theFAP-targeted single chain OX40 ligand trimer-containing Fc (kih) fusionantigen binding molecule. The polypeptide encoding the single chaintrimeric Ox40 ligand was subcloned in frame with the human IgG1 heavychain CH2 and CH3 domains on the knob (Merchant, Zhu et al. 1998), usinga linker (GSPGSSSSGS) of SEQ ID NO:57.

TABLE 27 Sequences of FAP(28H1)-targeted human OX40 ligand trimer-containing Fc (kih) fusion molecule (FAP single chain OX40Ltrimer) (Compound S10) SEQ ID NO: Description Sequence 87 nucleotideCAGGTGTCCCACAGATACCCCAGAATCCAGAGCATCAA sequence ofGGTGCAGTTCACCGAGTACAAGAAAGAGAAGGGCTTCA trimeric hu OX40LTCCTGACCAGCCAGAAAGAGGACGAGATCATGAAGGTG (51-183)-Fc knobCAGGACAACAGCGTGATCATCAACTGCGACGGCTTCTA chainCCTGATCAGCCTGAAGGGCTACTTCAGCCAGGAAGTGGACATCAGCCTGCACTACCAGAAGGACGAGGAACCCCTGTTCCAGCTGAAGAAAGTGCGGAGCGTGAACAGCCTGATGGTGGCCAGCCTGACCTACAAGGACAAGGTGTACCTGAACGTGACCACCGACAACACCAGCCTGGACGACTTCCACGTGAACGGCGGCGAGCTGATCCTGATTCACCAGAACCCCGGCGAGTTCTGCGTGCTGGGAGGCGGAGGATCTGGCGGAGGCGGATCTCAGGTGTCACACCGCTACCCCCGGATTCAGTCCATTAAGGTGCAGTTTACAGAGTATAAGAAAGAAAAAGGCTTTATTCTGACTTCCCAGAAAGAAGATGAGATTATGAAGGTGCAGGATAATTCTGTGATCATCAATTGTGATGGGTTTTATCTGATCTCCCTGAAAGGATACTTTAGTCAGGAAGTGGATATTTCTCTGCACTATCAGAAAGATGAAGAACCTCTGTTCCAGCTGAAAAAAGTGCGCTCCGTGAATTCTCTGATGGTGGCTTCCCTGACATACAAAGACAAAGTGTATCTGAATGTGACAACAGATAATACCTCCCTGGATGATTTCCATGTGAATGGGGGGGAACTGATTCTGATCCATCAGAACCCTGGGGAATTTTGTGTGCTGGGCGGAGGGGGAAGTGGCGGCGGAGGCAGTCAGGTGTCCCATCGGTATCCTAGAATCCAGTCTATCAAAGTGCAGTTTACTGAGTACAAAAAAGAGAAAGGATTCATTCTGACCTCTCAGAAAGAGGACGAAATTATGAAGGTGCAGGATAACAGTGTGATTATTAACTGTGATGGGTTCTACCTGATTTCTCTGAAGGGATATTTCAGTCAGGAAGTGGACATCTCACTGCATTACCAGAAGGATGAAGAACCACTGTTCCAGCTGAAGAAAGTGCGCTCTGTGAATAGCCTGATGGTGGCCTCTCTGACTTATAAGGATAAGGTGTACCTGAATGTGACAACTGACAATACTTCTCTGGACGACTTTCATGTGAACGGGGGAGAGCTGATTCTGATCCACCAGAATCCAGGCGAGTTTTGTGTGCTGGGAAGCCCCGGCAGCAGCAGCTCTGGATCCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCT CCGGGTAAA 67 nucleotideGAAGTGCAGCTGCTGGAATCCGGCGGAGGCCTGGTGCA sequence of anti-GCCTGGCGGATCTCTGAGACTGTCCTGCGCCGCCTCCGG FAP(28H1) FcCTTCACCTTCTCCTCCCACGCCATGTCCTGGGTCCGACA hole chainGGCTCCTGGCAAAGGCCTGGAATGGGTGTCCGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGACTCTGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGGCTGGCTGGGCAACTTCGACTACTGGGGACAGGGCACCCTGGTCACCGTGTCCAGCGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC GCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA68 nucleotide GAGATCGTGCTGACCCAGTCCCCCGGCACCCTGTCTCTG sequence of anti-AGCCCTGGCGAGAGAGCCACCCTGTCCTGCAGAGCCTC FAP(28H1) lightCCAGTCCGTGTCCCGGTCCTACCTCGCCTGGTATCAGCA chainGAAGCCCGGCCAGGCCCCTCGGCTGCTGATCATCGGCGCCTCTACCAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCCAGGTCATCCCTCCCACCTTTGGCCAGGGCACCAAGGTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCA CAAAGAGCTTCAACAGGGGAGAGTGT 20trimeric hu QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQDN OX40L (51-183)-SVIINCDGFYLISLKGYFSQEVDISLHYQKDEEPLFQLKKVR Fc knob chainSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLGGGGSGGGGSQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQDNSVIINCDGFYLISLKGYFSQEVDISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLGGGGSGGGGSQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQDNSVIINCDGFYLISLKGYFSQEVDISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLGSPGSSSSGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 18 anti-FAP(28H1)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQA Fc hole chainPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 19 anti-FAP (28H1)EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKP light chainGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC

The targeted single chain OX40 ligand trimer-containing Fc (kih) fusionantigen binding molecule encoding sequences were cloned into a plasmidvector driving expression of the insert from an MPSV promoter andcontaining a synthetic polyA sequence located at the 3′ end of the CDS.In addition, the vector contained an EBV OriP sequence for episomalmaintenance of the plasmid.

The targeted single chain trimeric OX40 ligand Fc (kih) fusion antigenbinding molecule was produced by co-transfecting HEK293-EBNA cells withthe mammalian expression vectors using polyethylenimine. The cells weretransfected with the corresponding expression vectors in a 1:1:1 ratio(“vector knob chain”: “vector hole chain”: “vector light chain”).

For production in 500 mL shake flasks, 400 million HEK293 EBNA cellswere seeded 24 hours before transfection. For transfection cells werecentrifuged for 5 minutes at 210 x g, and supernatant was replaced bypre-warmed CD CHO medium. Expression vectors were mixed in 20 mL CD CHOmedium to a final amount of 200 μg DNA. After addition of 540 μL PEI,the solution was vortexed for 15 seconds and incubated for 10 minutes atroom temperature. Afterwards, cells were mixed with the DNA/PEIsolution, transferred to a 500 mL shake flask and incubated for 3 hoursat 37° C. in an incubator with a 5% CO2 atmosphere. After theincubation, 160 mL F17 medium was added and cells were cultured for 24hours. One day after transfection 1 mM valproic acid and 7% Feed withsupplements were added. After culturing for 7 days, the supernatant wascollected by centrifugation for 15 minutes at 210 x g. The solution wassterile filtered (0.22 μm filter), supplemented with sodium azide to afinal concentration of 0.01% (w/v), and kept at 4° C.

The targeted single chain OX40 ligand trimer-containing Fc (kih) fusionantigen binding molecule was purified from cell culture supernatants byaffinity chromatography using Protein A, followed by size exclusionchromatography. For affinity chromatography, the supernatant was loadedon a HiTrap ProteinA HP column (CV=5 mL, GE Healthcare) equilibratedwith 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodiumchloride, 0.01% (v/v) Tween-20 containing buffer (pH 7.5). Unboundprotein was removed by washing with at least 10 column volumes of thesame buffer. The bound protein was eluted using a linear pH gradientover 20 column volumes to 100% of 20 mM sodium citrate, 0.5 M sodiumchloride, 0.01% (v/v) Tween-20 containing buffer (pH 2.5). The columnwas then washed with 10 column volumes of 20 mM sodium citrate, 0.5 Msodium chloride, 0.01% Tween-20 buffer (pH 2.5).

The pH of collected fractions was adjusted by adding 1/40 (v/v) of 2MTris, pH8.0. The protein was concentrated prior to loading on a HiLoadSuperdex 200 column (GE Healthcare) equilibrated with 20 mM Histidine,150 mM sodium chloride, 0.01% (v/v) Tween/20 solution of pH 6.0.

The protein concentration was determined by measuring the opticaldensity (OD) at 280 nm, using the molar extinction coefficientcalculated on the basis of the amino acid sequence. Purity and molecularweight of the targeted OX40 ligand trimer-containing Fc (kih) fusionantigen binding molecule was analyzed by SDS-PAGE in the presence andabsence of a reducing agent (5 mM 1,4-dithiotreitol) and staining withCoomassie SimpleBlueTM SafeStain (Invitrogen USA). The aggregate contentof samples was analyzed using a TSKgel G3000 SW XL analyticalsize-exclusion column (Tosoh) equilibrated in 25 mM K₂HPO₄, 125 mMsodium chloride, 200 mM L-arginine monohydrochloride, 0.02% (w/v) NaN₃,pH 6.7 running buffer at 25° C.

The targeted single chain OX40 ligand trimer-containing Fc (kih) fusionantigen binding molecule (Compound SC-10) was obtained with a yield of9.1 mg/ml and a final monomer content of 87% (SEC).

Example 7 Preparation and Purification of “Untargeted” Single Chain OX40Ligand Trimer-Containing Fc Fusion Antigen Binding Molecules

The “untargeted” construct was composed by the Ox40 ligand-Fc knob chaindescribed above, a DP47 germline control-Fc hole chain and a DP47 lightchain (FIG. 4B), which were co-transfected in a 1:1:1 ratio (“vectorknob chain”: “vector hole chain”: “vector light chain”).

Production and purification were performed as described above fortargeted single chain OX40 ligand trimer-containing Fc (kih) fusionantigen binding molecule. The variable region of heavy and light chainDNA sequences of the FAP binder, were replaced with those of a germlinecontrol (DP47) and subcloned in frame with either the constant heavychain of the hole or the constant light chain of human IgG1.

Table 28 shows, respectively, the cDNA and amino acid sequences of theDP47-containing 4-1BB ligand trimer-containing Fc (kih) fusion antigenbinding molecule.

TABLE 28 Sequences of DP47-containing single chain OX40 ligandtrimer-containing Fc (kih) fusion molecule (DP47 singlechain OX40L trimer) (Control 3) SEQ ID NO: Description Sequence 87nucleotide see Table 27 sequence of trimeric hu OX40L (51-183)-Fc knobchain 69 nucleotide GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACsequence of DP47 AGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCC Fc hole chainGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGCAGCGGATTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCC TCTCCCTGTCTCCGGGTAAA 70 nucleotideGAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTT sequence of DP47GTCTCCAGGGGAAAGAGCCACCCTCTCTTGCAGGGCCA light chainGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGCTGACGTTCGGCCAGGGGACCAAAGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAG AGTGT 20 trimeric hu see Table 27OX40L (51-183)- Fc knob chain 71 DP47 Fc holeEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQ chainAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK 72DP47 light chain EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC

The DP47-containing single chain OX40 ligand trimer-containing Fc (kih)fusion antigen binding molecule (Control 3) was obtained in a yield of 7mg/ml and a final monomer content of 100% (SEC).

Example 8 Preparation, Purification and Characterization of OX40

DNA sequences encoding the ectodomains of human, mouse or cynomolgusOX40 (Table 11) were subcloned in frame with the human IgG1 heavy chainCH2 and CH3 domains on the knob (Merchant et al., 1998). An AcTEVprotease cleavage site was introduced between an antigen ectodomain andthe Fc of human IgG1. An Avi tag for directed biotinylation wasintroduced at the C-terminus of the antigen-Fc knob. Combination of theantigen-Fc knob chain containing the S354C/T366W mutations, with a Fchole chain containing the Y349C/T366S/L368A/Y407V mutations allowsgeneration of a heterodimer which includes a single copy of the OX40ectodomain containing chain, thus creating a monomeric form of Fc-linkedantigen (FIG. 4C). Table 29 shows the amino acid sequences of thevarious OX40 ectodomains. Table 30 shows the cDNA and amino acidsequences of monomeric antigen Fc(kih) fusion molecules as depicted inFIG. 4C.

TABLE 29 Amino acid numbering of antigen ectodomains (ECD) and theirorigin SEQ ID NO: Construct Origin ECD 88 human Ox40 ECD Synthetizedaccording to P43489 aa 29-214 89 cynomolgus Ox40 isolated fromcynomolgus blood aa 29-214 ECD 90 murine Ox40 ECD Synthetized accordingto P47741 aa 10-211

TABLE 30 cDNA and Amino acid sequences of monomeric antigen Fc(kih)fusion molecules (produced by combination of one Fc hole chainwith one antigen Fc knob chain) SEQ ID NO: Antigen Sequence 76Nucleotide GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGA sequenceACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAA Fc hole chainACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGC AGAAGAGCCTCTCCCTGTCTCCGGGTAAA 91Nucleotide CTGCACTGCGTGGGCGACACCTACCCCAGCAACGACCG sequenceGTGCTGCCACGAGTGCAGACCCGGCAACGGCATGGTGT human OX40CCCGGTGCAGCCGGTCCCAGAACACCGTGTGCAGACCTT antigen Fc knobGCGGCCCTGGCTTCTACAACGACGTGGTGTCCAGCAAGC chainCCTGCAAGCCTTGTACCTGGTGCAACCTGCGGAGCGGCAGCGAGCGGAAGCAGCTGTGTACCGCCACCCAGGATACCGTGTGCCGGTGTAGAGCCGGCACCCAGCCCCTGGACAGCTACAAACCCGGCGTGGACTGCGCCCCTTGCCCTCCTGGCCACTTCAGCCCTGGCGACAACCAGGCCTGCAAGCCTTGGACCAACTGCACCCTGGCCGGCAAGCACACCCTGCAGCCCGCCAGCAATAGCAGCGACGCCATCTGCGAGGACCGGGATCCTCCTGCCACCCAGCCTCAGGAAACCCAGGGCCCTCCCGCCAGACCCATCACCGTGCAGCCTACAGAGGCCTGGCCCAGAACCAGCCAGGGGCCTAGCACCAGACCCGTGGAAGTGCCTGGCGGCAGAGCCGTCGACGAACAGTTATATTTTCAGGGCGGCTCACCCAAATCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGAACGACATCTTC GAGGCCCAGAAGATTGAATGGCACGAG 92Nucleotide CTCCACTGTGTCGGGGACACCTACCCCAGCAACGACCG sequenceGTGCTGTCAGGAGTGCAGGCCAGGCAACGGGATGGTGA cynomolgusGCCGCTGCAACCGCTCCCAGAACACGGTGTGCCGTCCGT OX40 antigenGCGGGCCCGGCTTCTACAACGACGTGGTCAGCGCCAAG Fc knob chainCCCTGCAAGGCCTGCACATGGTGCAACCTCAGAAGTGGGAGTGAGCGGAAACAGCCGTGCACGGCCACACAGGACACAGTCTGCCGCTGCCGGGCGGGCACCCAGCCCCTGGACAGCTACAAGCCTGGAGTTGACTGTGCCCCCTGCCCTCCAGGGCACTTCTCCCCGGGCGACAACCAGGCCTGCAAGCCCTGGACCAACTGCACCTTGGCCGGGAAGCACACCCTGCAGCCAGCCAGCAATAGCTCGGACGCCATCTGTGAGGACAGGGACCCCCCACCCACACAGCCCCAGGAGACCCAGGGCCCCCCGGCCAGGCCCACCACTGTCCAGCCCACTGAAGCCTGGCCCAGAACCTCACAGAGACCCTCCACCCGGCCCGTGGAGGTCCCCAGGGGCCCTGCGGTCGACGAACAGTTATATTTTCAGGGCGGCTCACCCAAATCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGAACGACATC TTCGAGGCCCAGAAGATTGAATGGCACGAG93 murine OX40 GTGACCGCCAGACGGCTGAACTGCGTGAAGCACACCTA antigen Fc knobCCCCAGCGGCCACAAGTGCTGCAGAGAGTGCCAGCCCG chainGCCACGGCATGGTGTCCAGATGCGACCACACACGGGACACCCTGTGCCACCCTTGCGAGACAGGCTTCTACAACGAGGCCGTGAACTACGATACCTGCAAGCAGTGCACCCAGTGCAACCACAGAAGCGGCAGCGAGCTGAAGCAGAACTGCACCCCCACCCAGGATACCGTGTGCAGATGCAGACCCGGCACCCAGCCCAGACAGGACAGCGGCTACAAGCTGGGCGTGGACTGCGTGCCCTGCCCTCCTGGCCACTTCAGCCCCGGCAACAACCAGGCCTGCAAGCCCTGGACCAACTGCACCCTGAGCGGCAAGCAGACCAGACACCCCGCCAGCGACAGCCTGGATGCCGTGTGCGAGGACAGAAGCCTGCTGGCCACCCTGCTGTGGGAGACACAGCGGCCCACCTTCAGACCCACCACCGTGCAGAGCACCACCGTGTGGCCCAGAACCAGCGAGCTGCCCAGTCCTCCTACCCTCGTGACACCTGAGGGCCCCGTCGACGAACAGTTATATTTTCAGGGCGGCTCACCCAAATCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGAACGACATCTTCGAGGCCCAGAAGATTG AATGGCACGAG 80 Fc hole chainDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK 94human OX40 LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPC antigen Fc knobGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVC chainRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKI EWHE 95 cynomolgusLHCVGDTYPSNDRCCQECRPGNGMVSRCNRSQNTVCRPC OX40 antigenGPGFYNDVVSAKPCKACTWCNLRSGSERKQPCTATQDTV Fc knob chainCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPPTQPQETQGPPARPTTVQPTEAWPRTSQRPSTRPVEVPRGPAVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQ KIEWHE 96 murine OX40VTARRLNCVKHTYPSGHKCCRECQPGHGMVSRCDHTRDT antigen Fc knobLCHPCETGFYNEAVNYDTCKQCTQCNHRSGSELKQNCTPT chainQDTVCRCRPGTQPRQDSGYKLGVDCVPCPPGHFSPGNNQACKPWTNCTLSGKQTRHPASDSLDAVCEDRSLLATLLWETQRPTFRPTTVQSTTVWPRTSELPSPPTLVTPEGPVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIF EAQKIEWHE

All OX40-Fc-fusion encoding sequences were cloned into a plasmid vectordriving expression of the insert from an MPSV promoter and containing asynthetic polyA signal sequence located at the 3′ end of the CDS. Inaddition, the vector contained an EBV OriP sequence for episomalmaintenance of the plasmid.

For preparation of the biotinylated monomeric antigen/Fc fusionmolecules, exponentially growing suspension HEK293 EBNA cells wereco-transfected with three vectors encoding the two components of fusionprotein (knob and hole chains) as well as BirA, an enzyme necessary forthe biotinylation reaction. The corresponding vectors were used at a2:1:0.05 ratio (“antigen ECD-AcTEV-Fc knob”: “Fc hole”: “BirA”).

For protein production in 500 ml shake flasks, 400 million HEK293 EBNAcells were seeded 24 hours before transfection. For transfection cellswere centrifuged for 5 minutes at 210 g, and supernatant was replaced bypre-warmed CD CHO medium. Expression vectors were resuspended in 20 mLof CD CHO medium containing 200 μg of vector DNA. After addition of 540μL of polyethylenimine (PEI), the solution was vortexed for 15 secondsand incubated for 10 minutes at room temperature. Afterwards, cells weremixed with the DNA/PEI solution, transferred to a 500 mL shake flask andincubated for 3 hours at 37° C. in an incubator with a 5% CO₂atmosphere. After the incubation, 160 mL of F17 medium was added andcells were cultured for 24 hours. One day after transfection, 1 mMvalproic acid and 7% Feed were added to the culture. After 7 days ofculturing, the cell supernatant was collected by spinning down cells for15 min at 210 g. The solution was sterile filtered (0.22 μm filter),supplemented with sodium azide to a final concentration of 0.01% (w/v),and kept at 4° C.

Secreted proteins were purified from cell culture supernatants byaffinity chromatography using Protein A, followed by size exclusionchromatography. For affinity chromatography, the supernatant was loadedon a HiTrap ProteinA HP column (CV=5 mL, GE Healthcare) and equilibratedwith 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodiumchloride, 0.01% (v/v) Tween-20 containing buffer (pH 7.5). Unboundprotein was removed by washing with at least 10 column volumes of thesame buffer. The bound protein was eluted using a linear pH gradientover 12 column volumes to 100% of 20 mM sodium citrate, 0.5 M sodiumchloride, 0.01% (v/v) Tween-20 containing buffer (pH 2.5). The columnwas then washed with 10 column volumes of 20 mM sodium citrate, 0.5 Msodium chloride, 0.01% Tween-20 buffer (pH 2.5).

The pH of the collected fractions was adjusted by adding 1/40 (v/v) of2M Tris, pH8.0. The protein was concentrated and filtered prior toloading on a HiLoad Superdex 200 column (GE Healthcare) equilibratedwith 2 mM MOPS, 150 mM sodium chloride, 0.02% (w/v) sodium azidesolution of pH 7.4.

For affinity determination to the human receptor, the ectodomain ofhuman OX40 was also subcloned in frame with an avi (GLNDIFEAQKIEWHE, SEQID NO:84) and a hexahistidine tag. The sequences are shown in Table 31.

Protein production was performed as described above for the Fc-fusionprotein. Secreted proteins were purified from cell culture supernatantsby chelating chromatography, followed by size exclusion chromatography.The first chromatographic step was performed on a NiNTA SuperflowCartridge (5m1, Qiagen) equilibrated with a solution of 20 mM sodiumphosphate and 500 nM sodium chloride, pH 7.4. Elution was performed byapplying a gradient over 12 column volume from 5% to 45% of elutionbuffer containing 20 mM sodium phosphate, 500 nM sodium chloride and 500mM Imidazole (pH 7.4). The protein was concentrated and filtered priorto loading on a HiLoad Superdex 75 column (GE Healthcare) equilibratedwith a solution of 2 mM MOPS, 150 mM sodium chloride and 0.02% (w/v)sodium azide (pH 7.4).

TABLE 31 Sequences of monomeric human OX40 His molecule SEQ ID NO:antigen Sequence 97 nucleotide CTGCACTGCGTGGGCGACACCTACCCCAGCAACGACCGGTsequence GCTGCCACGAGTGCAGACCCGGCAACGGCATGGTGTCCCG humanGTGCAGCCGGTCCCAGAACACCGTGTGCAGACCTTGCGGC OX40 HisCCTGGCTTCTACAACGACGTGGTGTCCAGCAAGCCCTGCAAGCCTTGTACCTGGTGCAACCTGCGGAGCGGCAGCGAGCGGAAGCAGCTGTGTACCGCCACCCAGGATACCGTGTGCCGGTGTAGAGCCGGCACCCAGCCCCTGGACAGCTACAAACCCGGCGTGGACTGCGCCCCTTGCCCTCCTGGCCACTTCAGCCCTGGCGACAACCAGGCCTGCAAGCCTTGGACCAACTGCACCCTGGCCGGCAAGCACACCCTGCAGCCCGCCAGCAATAGCAGCGACGCCATCTGCGAGGACCGGGATCCTCCTGCCACCCAGCCTCAGGAAACCCAGGGCCCTCCCGCCAGACCCATCACCGTGCAGCCTACAGAGGCCTGGCCCAGAACCAGCCAGGGGCCTAGCACCAGACCCGTGGAAGTGCCTGGCGGCAGAGCCGTCGACGAACAGTTATATTTTCAGGGCGGCTCAGGCCTGAACGACATCTTCGAGGCCCAGAAGATCGAGTGGCACGAGGCTCGA GCTCACCACCATCACCATCAC 98 humanLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGP OX40 HisGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVDEQLYFQGGSGLNDIFEAQKIE WHEARAHHHHHH

Example 9 Biochemical Characterization of FAP-Targeted Single Chain OX40Ligand Trimer-Containing Fc Fusion Antigen Binding Molecule by SurfacePlasmon Resonance

Binding of FAP-targeted single chain OX40 ligand trimer-containing Fc(kih) fusion antigen binding molecule to recombinant OX40 was assessedby surface plasmon resonance (SPR). All SPR experiments were performedon a Biacore T100 at 25° C. with HBS-EP as a running buffer (0.01 MHEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore,Freiburg/Germany).

The avidity of the interaction between the FAP-targeted or untargetedOX40 ligand trimer-containing Fc (kih) fusion antigen binding moleculesand recombinant OX40 (human, cyno and murine) was determined asillustrated in FIG. 8A. The data demonstrated that both variants of OX40ligand trimer-containing Fc (kih) fusion antigen binding molecules (FAPsingle chain OX40 trimer (compound S10 in FIGS. 8A-D, andDP47-containing single chain OX40 trimer (Control 3) in FIGS. 9A-C) bindwith comparable avidities to human and cynomolgus OX40 but negligibly tothe mouse homolog.

Recombinant biotinylated human, cynomolgus and murine OX40 Fc(kih)fusion molecules were directly coupled on a SA chip using the standardcoupling instruction (Biacore, Freiburg/Germany). The immobilizationlevel was about 600 resonance units (RU). FAP-targeted OX40 ligandtrimer-containing Fc (kih) fusion antigen binding molecule, or theDP47-containing control, was passed at a concentration range from 0.2 to500 nM with a flow of 30 μL/minutes through the flow cells over 120seconds. The dissociation was monitored for 220 seconds. Bulk refractiveindex differences were corrected by subtracting the response obtained ona reference empty flow cell.

For affinity measurement (FIGS. 10A-C), direct coupling of around 7200resonance units (RU) of the anti-human Fc specific antibody wasperformed on a CMS chip at pH 5.0 using the standard amine coupling kit(GE Healthcare). FAP-targeted or untargeted single chain trimeric Ox40ligand Fc(kih) fusion molecule at 200 nM was captured with a flow rateof 30 μl/min for 60 sec on flow cell 2. A dilution series (31-2000 nM)of huOX40 avi His was passed on both flow cells at 30 μl/min for 120 secto record the association phase. The dissociation phase was monitoredfor 180 s and triggered by switching from the sample solution to HBS-EP.The chip surface was regenerated after every cycle using a doubleinjection of 60 sec 3M MgCl₂. Bulk refractive index differences werecorrected for by subtracting the response obtained on the reference flowcell 1. For the interaction between targeted single chain trimeric OX40ligand Fc(kih) fusion molecule and hu0x40 avi His, the affinityconstants were derived from the rate constants by fitting to a 1:1Langmuir binding using the Biaeval software (GE Healthcare).Dissociation constants can be found in Table 32.

TABLE 32 Fittings to 1:1 Langmuir binding and Affinity constants LigandAnalyte ka (1/Ms) kd (1/s) KD (M) FAP single chain Hu OX40 1.8E+051.3E−01 7.5E−07 OX40 trimer avi His Compound S10

Example 10 Functional Characterization of the Targeted 4-1BB Ligand orOX40 Ligand Trimer-Containing Fc Fusion Antigen Binding Molecules

10.1. Binding on Naïve Versus Activated Human PMBCs of the FAP-Targeted4-1BB Ligand or OX40 Ligand Trimer-Containing Fc (kih) Fusion AntigenBinding Molecules

Buffy coats were obtained from the Zurich blood donation center. Toisolate fresh peripheral blood mononuclear cells (PBMCs) the buffy coatwas diluted with the same volume of DPBS (Gibco by Life Technologies,Cat. No. 14190 326). 50 mL polypropylene centrifuge tubes (TPP, Cat.-No.91050) were supplied with 15 mL Histopaque 1077 (SIGMA Life Science,Cat.-No. 10771, polysucrose and sodium diatrizoate, adjusted to adensity of 1.077 g/mL) and the buffy coat solution were layered abovethe Histopaque 1077. The tubes were centrifuged for 30 min at 450 x g.PBMCs were collected from the interface, washed three times with DPBSand resuspended in T cell medium consisting of RPMI 1640 medium (Gibcoby Life Technology, Cat. No. 42401-042) supplied with 10% Fetal BovineSerum (FBS, Gibco by Life Technology, Cat. No. 16000-044, Lot 941273,gamma-irradiated, mycoplasma-free and heat inactivated at 56° C. for 35min), 1% (v/v) GlutaMAX I (GIBCO by Life Technologies, Cat. No. 35050038), 1 mM Sodium-Pyruvat (SIGMA, Cat. No. S8636), 1% (v/v) MEMnon-essential amino acids (SIGMA, Cat.-No. M7145) and 50 μMβ-Mercaptoethanol (SIGMA, M3148).

PBMCs were used directly after isolation (naïve, resting PBMCs) or theywere stimulated to induce 4-1BB and OX40 expression on the cell surfaceof T cells (activated PBMCs) as following: naïve PBMCs were cultured forfour days in T cell medium supplemented with 200 U/mL Proleukin and 2ug/mL PHA-L in a 6-well tissue culture plate. For reactivation cellswere further activated for 1 day on 6-well tissue culture plate coatedwith 10 ug/mL anti-human CD3 (clone OKT3) and 2 ug/mL anti-human CD28(clone CD28.2) in the T cell medium supplemented with 200 U/mL Proleukinat 37° C. and 5% CO₂.

For detection of 4-1BB binding, 0.1×106 naïve or activated PBMCs wereadded to each well of a round-bottom suspension cell 96-well plates(greiner bio-one, cellstar, Cat. No. 650185). For detection of OX40,naïve human PBMC and activated human PBMC were mixed. To enablediscrimination of naïve and activated human PBMC naïve cells werelabeled prior to the binding assay using the eFluor670 cellproliferation dye (eBioscience, Cat.-No.65-0840-85).

For eFluor670 cell proliferation dye labelling, cells were harvested,washed with pre-warmed (37° C.) DPBS and adjusted to a cell density of1×10⁷ cells/mL in DPBS. eFluor670 cell proliferation dye (eBioscience,Cat.-No.65-0840-85) was added to naïve human PBMC at a finalconcentration of 2.5 mM and a final cell density of 0.5×10⁷ cells/mL inDPBS. Cells were then incubated for 10 min at room temperature in thedark. To stop labeling reaction, 2 mL FBS were added and cells werewashed three times with the T cell medium. A 1 to 1 mixture of 1×10⁵naive, eFluor670 labeled human PBMC and unlabeled activated human PBMCwere then added to each well of a round-bottom suspension cell 96-wellplates (greiner bio-one, cellstar, Cat. No. 650185).

Plates were centrifuged 4 minutes at 400×g and at 4° C. and supernatantwas flicked off. Cells were washed once with 200 μL 4° C. cold FACSbuffer (DPBS supplied with 2% FBS, 5 mM EDTA pH8 (Amresco, Cat. No.E177) and 7.5 mM Sodium azide (Sigma-Aldrich Cat. No. S2002)). Cellswere incubated in 50 μL/well of 4° C. cold FACS containing titratedFAP-targeted or DP47 untargeted single chain 4-1BB or OX40 ligandtrimer-containing Fc fusion antigen binding molecules for 120 minutes at4° C. Plates were washed four times with 200 μL/well 4° C. FACS buffer.

For detection of 4-1BB binding, cells were resuspended in 50 μL/well 4°C. cold FACS buffer containing 0.125 μg/mL anti-human CD4-BV421 (cloneRPA-T4, mouse IgG1 k, BioLegend, Cat.-No. 300532), 20 μL/mL anti-humanCD3-PerCP/Cy5.5 (clone SK7, mouse IgG1k, BD Bioscience, Cat.-No.332771), 0.25 μg/mL anti-human CD8a-APC (clone RPA-T8, mouse IgG1k,BioLegend, Cat.-No. 301014), 0.25 μg/mL anti-human CD56-AF488 (cloneHCD56, mouse IgGlk, BD Pharmingen, Cat.-No. 318310), 2.5 μg/mLanti-human Fcy-specific goat IgG F(ab)₂ fragment-PE (JacksonImmunoresearch, Cat. No. 109 116 098 or 109-116-170) and incubated for30 minutes at 4° C. in the dark.

For detection of OX40 binding, cells were resuspended in 50 μL/well 4°C. cold FACS buffer containing 0.255 μg/mL anti-human CD4-BV421 (cloneRPA-T4, mouse IgG1 k, BioLegend, Cat.-No. 300532), 0.250 μg/mLanti-human CD8-BV711 (clone RPa-T8, mouse IgG1k, BioLegend, Cat.-No.3010441), 0.250 μg/mL anti-human CD45-PerCP Cy5.5 (clone HI30, mouseIgGlk, BioLegend, Cat.-No. 304028), and 30 μg/mL Fluoresceinisothiocyanate (FITC)-conjugated AffiniPure anti-human IgGFcy-fragment-specific goat IgG F(ab′)₂ fragment (Jackson ImmunoResearch,Cat. No. 109 096 098) and incubated for 30 minutes at 4° C. in the dark.

Plates where then washed twice with 200 μL/well 4° C. FACS buffer andcells were finally resuspended in 80 μL/well FACS-buffer containing 0.2μg/mL DAPI (Santa Cruz Biotec, Cat. No. Sc-3598) and acquired the sameday using 5-laser LSR-Fortessa (BD Bioscience with DIVA software).

In some cases, cells were fixed and acquired the next day. For thismethod cells were stained additionally prior to incubation with primaryantibody with 100 μL/well DPBS containing 1:1000 diluted LIVE/DEADFixable Blue Dead Cell Stain Kit, for UV excitation (Life Technologies,Molecular Probes, Cat. No. L-23105) for 30 minutes at 4° C. in the dark.After incubation with a secondary antibody cocktail, plates were washedtwice with 200 μL/well 4° C. FACS buffer, fixed with 50 DPBS containing1% Formaldehyde (Sigma, HT501320-9.5L) and incubated overnight at 4° C.For flow cytomtric analysis cells were resuspended in 80 μl/well FACSbuffer.

As shown in FIGS. 11A to 11D and FIGS. 12A to 12D, neither FAP-targetednor DP47-containing single chain 4-1BBL and OX40L trimer-containing Fc(kih) fusion antigen binding molecules did bind to resting human CD4⁺ Tcells or CD8⁺ T cells. In contrast, all constructs bound strongly toactivated CD8⁺ or CD4⁺ T cells. The single chain 4-1BBLtrimer-containing molecules showed approximately 10 fold lower bindingto CD4+ as compared to CD8+ T-cells. In contrast, the single chain Ox40Ltrimer-containing molecules showed approximately 4 fold higher intensityof specific fluorescence upon binding to CD4⁺ as compared to CD8⁺ Tcells. These differences concur very well with 10-20 fold higer levelsof 4-1BB expression on CD8⁺ human T-cells as compared to CD4⁺ T-cellswhile the latter express approximately 4 fold higher levels of OX40 ascompared to the CD8⁺ subset. As shown in FIGS. 11E to 11H, monovalentand bivalent FAP (28H1 or 4B9)-targeted or CEA (sm9b)-targeted orDP47-untargeted single chain trimeric 4-1BB ligand Fc (kih) fusionmolecules did not bind to resting human CD4⁺ or CD8⁺ T cells butstrongly to activated CD8⁺ T cells and to a less extend to activatedCD4⁺ T cells. As can be seen in FIGS. 11J to 11M, the binding wasindependent of targeting or bivalency, e.g. independent of the fusionlocation of the single chain trimeric 4-1BB ligand.

10.2. Binding to FAP-Expressing Tumor Cells

For binding assays on FAP expressing cells, human fibroblast activatingprotein (huFAP) expressing cells NIH/3T3-huFAP clone 39 or WM266-4 cells(ATCC, Cat. No. CRL-1676) were used. The NIH/3T3-huFAP clone 39 wasgenerated by the transfection of the mouse embryonic fibroblast NIH/3T3cell line (ATCC, Cat. No. CRL-1658) with the expression vector pETR4921to express huFAP under 1.5 μg/mL Puromycin selection.

0.1×10⁶ NIH/3T3-huFAP clone 39 (4-1BBL) or 33 10⁴ WM266-4 cells (OX40L)in DPBS were added to each well of round-bottom suspension cell 96-wellplates. Plates were centrifuged 4 minutes, 400×g at 4° C. andsupernatants were flicked off. Cells were washed once with 200 μL DPBSand pellets were resuspended by a short and gentle vortex. For bindinganalysis of the 4-1BBL trimer-containing Fc (kih) fusion antigen bindingmolecule, 100 μL/well of 4° C. cold DPBS buffer containing 1:5000diluted Fixable Viability Dye eFluor 450 (eBioscience, Cat. No. 65 086318) were added and plates were incubated for 30 minutes at 4° C. Cellswere washed once with 200 μL 4° C. cold DPBS buffer. All samples wereresuspended in 50 μL/well of 4° C. cold FACS buffer containing theFAP-targeted or DP47-containing single chain 4-1BBL or OX40Ltrimer-containing molecules at the indicated range of cocentrations andincubated for 1 hour at 4° C. Afterwards the cells were washed fivetimes with 200 μL 4° C. FACS buffer and resuspended by a short vortex.Cells were further stained with 50 μL/well of 4° C. cold FACS buffercontaining 30 μg/mL Fluorescein isothiocyanate (FITC)-conjugatedAffiniPure anti-human IgG Fcγ-fragment-specific goat IgG F(ab′)₂fragment (Jackson ImmunoResearch, Cat. No. 109 096 098) for 30 minutesat 4° C. Cells were washed twice with 200 μL 4° C. FACS buffer. Cellsstained with OX40L trimer-containing molecule were finally resuspendedin 80 μL/well FACS-buffer containing 0.2 μg/mL DAPI (Santa Cruz Biotec,Cat. No. Sc-3598) and acquired the same day using 5-laser LSR-Fortessa(BD Bioscience with DIVA software).

Cells stained with 4-1BBL trimer-containing antigen binding moleculesand cells were fixed by resuspension in 50 DPBS containing 1%Formaldehyde. The next day cells were resuspended in 100 μL FACS-bufferand acquired using 5-laser LSR-Fortessa (BD Bioscience with DIVAsoftware).

As shown in FIGS. 13A-E, the FAP-targeted molecules containing eitherthe single chain trimeric 4-1BBL (compound 51, FIG. 13A) or OX40L(compound S10, FIG. 13B) efficiently bound to human FAP-expressig cells.In contrast, their counterparts containing the DP47 antibody (Control 1and 3) showed no such binding (FIGS. 13A and 13B). Therefore, onlyFAP-targeted single chain trimeric 4-1BBL or OX40L containing moleculesshowed tumor-targeting properties. As shown, in FIGS. 13C and 13D,monovalent and bivalent FAP (28H1 or 4B9)-targeted, but notDP47-untargeted single chain trimeric 4-1BBL Fc (kih) fusion antigenbinding molecules can bind to FAP-expressing human melanoma cell linesMV-3 and WM-266-4. FAP-binding clone 4B9 shows the strongest bindingwhereas clone 28H1 shows a lower affinity on FAP-expressing tumor cells.Due to the lower affinity, differences between the monovalent FAP(28H1)-targeted single chain trimeric 4-1BBL (kih) fusion molecule andthe bivalent binding FAP (28H1)-targeted hu IgG1 P329G LALA controlmolecule become more obvious. The bivalent binding FAP(28H1)-targeted huIgG1 P329G LALA control molecule shows a lower MFI (bivalent occupancyof the FAP molecules) and lower EC₅₀ value.

10.3. Binding to CEA-Expressing Tumor Cells

For binding assays on CEA-expressing tumor cells, the following humanCEA-expressing tumor cell line was used: human gastric cancer (stomach)cell line received from a liver metastasis MKN45 (DMSZ, No. ACC 409).The assays were performed as described for the FAP-expressing MV-3 andWM-266-4 tumor cell lines in Example 10.2.

As shown in FIG. 13E, monovalent and bivalent CEA (sm9b)-targeted butnot DP47-untargeted single chain 4-1BB ligand trimer-containing Fc (kih)fusion antigen binding molecules can bind to CEA-expressing humangastric cancer cell line MKN45. Differences between the monovalent CEA(sm9b)-targeted single chain trimeric 4-1BBL Fc (kih) fusion molecule(Compound S5) and the bivalent CEA (sm9b)-targeted single chain trimeric4-1BBL Fc (kih) fusion molecule (Compound S4) is shown in a lower MFI(bivalent occupancy of the CEA molecules).

10.4. Binding to Activated Mouse Splenocytes Expressing Mouse 4-1BB

Mouse spleens were collected in 3 mL PBS and a single cell suspensionwas generated using the gentle MACS tubes (Miltenyi Biotec Cat.-No.130-096-334) and gentle MACS Octo Dissociator (Miltenyi Biotec).Afterwards splenocytes were filtered through a 70 μm pre-separationfilters (Miltenyi Biotec Cat.-No. 130-041-407) and centrifuged for 7 minat 350×g and 4° C. Supernatants were aspirated and cells wereresuspended in RPMI 1640 medium supplied with 10% (v/v) FBS, 1% (v/v)GlutaMAX I, 1 mM Sodium-Pyruvate, 1% (v/v) MEM non-essential aminoacids, 50 μM β-Mercaptoethanol and 10% Penicillin-Streptomycin (SIGMA,Cat.-No. P4333). 10⁶ cells/mL were cultured for 2 days in a 6-welltissue culture plate coated with 10 μg/mL anti-mouse CD3c Armenianhamster IgG (clone 145-2C11, BioLegend, Cat.-No. 100331) and 2 μg/mLanti-mouse CD28 Syrian hamster IgG (clone 37.51, BioLegend, Cat.-No.102102). Activated mouse splenocytes were harvested, washed in DPBS,counted and 0.1×10⁶ cells were transferred to each well of a 96 U-bottomnon-tissue culture treated well plate. Supernatant was removed and cellswere stained in 100 uL/well DPBS containing 1:5000 diluted FixableViability Dye eF660 (Bioscience, Cat-No. 65-0864-18) for 30 min at 4° C.Cells were washed with DPBS and stained in 50 uL FACS buffer containingdifferent concentration of FAP-targeted or DP47-targeted single chaintrimeric 4-1BBL antigen binding molecule or anti-mouse CD137 human IgG1P329G LALA mAb (clone Lob.12.3). Cells were incubated for 60 min at 4°C. Then cells were washed four times with FACS buffer and stained in 50μL/well FACS buffer containing 10 μg/mL purified anti-mouse CD16/CD32rat IgG-Fc-Block (BD Pharmingen, Cat.-No. 553142 clone 2.4G2), 5 μg/mLanti-mouse CD8b rat IgG2bκ-FITC (BioLegend, Cat.-No. 126606, cloneYTS156.7.7), 0.67 μg/mL anti-mouse CD3 rat IgG2bκ-APC-Cy7 (BioLegend,Cat.-No. 100222, clone 17A2), 0.67 μg/mL anti-mouse CD4 ratIgG2bκ-PE-Cy7 (BioLegend, Cat.-No. 100422, clone GK1.5), 2 μg/mLanti-mouse NK1.1 Mouse (C3H x BALB/c) IgG2aκ-PerCp-Cy5.5 (BioLegend,Cat.-No. 108728, clone PK136) and 10 μg/mL PE-conjugated AffiniPurepolyclonal F(ab′)₂ Fragment goat anti-human IgG, Fcγ fragment specific,minimal cross-reactive to Bovine mouse and rabbit serum proteins(Jackson ImmunoResearch, Cat.-No. 109-116-170) for 30 min at 4° C. Cellswere washed twice with 200 μL/well cold FACS buffer, fixed with 50μL/well DPBS containing 1% Formaldehyde and stored overnight at 4° C.The next day cells were resuspended in FACS-buffer and acquired using a3-laser Canto II flow cytometer (BD Bioscience with DIVA software).

As shown in FIGS. 14A-B, the FAP-targeted or DP47-containing singlechain trimeric human 4-1BBL molecules (Compound S1 and Control 1) do notbind to mouse 4-1BB. Therefore their activity cannot be tested in immunecompetent mice. For in vivo mode of action studies either humanizedmouse models or surrogates containing single chain trimeric mouse 4-1BBLhave to be used.

Example 11 Biological Activity of the Targeted 4-1BB Ligand or OX40Ligand Trimer-Containing Fc Fusion Antigen Binding Molecules

11.1. NFκB Activation in HeLa Cells Expressing Human 4-1BB or OX40

11.1.1. Generation of HeLa Cells Expressing Human 4-1BB and LuciferaseUnder Control of an NFκB Enhancer Region

The human-papilloma-virus-18-induced cervix carcinoma cell line HeLa(ATCC, Cat. No. CCL-2) was transduced with the expression vectorpETR10829, which contains the human sequence of 4-1BB (Uniprot accessionQ07011, SEQ ID NO:99) under control of the CMV-promoter and thePuromycin resistance gene. Cells were cultured in DMEM-medium suppliedwith 10% FBS, 1% GlutaMAX-I and 3 μg/mL Puromycin (InvivoGen, Cat-No.ant-pr). 4-1BB-transduced HeLa cells were tested for 4-1BB expression byflow cytometry: 0.2×10⁶ living cells were resuspended in 100 μL FACSbuffer containing 0.1 μg PerCP/Cy5.5 conjugated anti-human 4-1BB mouseIgG1κ clone 4B4-1 (BioLegend Cat. No. 309814) or its isotype control(PerCP/Cy5.5 conjugated mouse IgG1κ isotype control antibody clone MOPC21, BioLegend Cat. No. 400150) and incubated for 30 minutes at 4° C.Cells were washed twice with FACS buffer, resuspended in 300 μL FACSbuffer containing 0.06 μg DAPI and acquired using 5-laser LSR-Fortessathe same day. Single clones were generated by limited dilution. To thisend, living human-4-1BB transduced HeLa cells were resuspended in mediumto a density of 10, 5 and 2.5 cells/mL and 200 μL of these cellsuspensions were transferred to round bottom tissue-culture treated96-well plates (6 plates/cell concentration, TPP Cat. No. 92697). Singleclones were harvested, expanded and tested for 4-1BB expression asdescribed above. The clone with the highest expression of 4-1BB (clone5) was chosen for further transfection with the NF-κB-luciferaseexpression-vector 5495p Tranlucent HygB, which was generated at Roche.The expression-vector 5495p Tranlucent HygB combines a Hygromycin Bresistance into NF-κB(1) Luciferase Reporter Vector (Panomics, Cat. No.LR0051). Human-4-1BB HeLa clone 5 cells were cultured to a confluency of70 percent. 50 μg (40 μL) of linearized (restriction enzymes AseI andSalI) 5495p Tranlucent HygB expression vector DNA were added to asterile 0.4 cm Gene Pulser/MicroPulser Cuvette (Biorad, Cat.-No,165-2081). 2.5×10⁶ human-4-1BB HeLa clone 5 cells in 400 μlsupplement-free DMEM were added and mixed carefully with the plasmidsolution. The cuvette was implemented into the Gene Pulser Xcell totalsystem (Biorad, Cat No. 165 2660) and an electroporation transfectionwas performed with following settings: Exponential pulse, capacitance500 μF, voltage 160 V, resistance ∞. Immediately after the pulsetransfected cells were transferred to a tissue culture flask 75 cm²(TPP, Cat. No. 90075) with 15 mL 37° C. warm DMEM-Medium supplied with10% FBS and 1% GlutaMAX I. 24 hours after seeding the selection mediawas added containing 3 μg/mL Puromycin and 200 μg/mL Hygromycin B(Roche, Cat. No. 10843555001). Afterwards surviving cells were expandedand limited dilution was performed as described above to generate singleclones.

Clones were tested for 4-1BB expression as described above and forNF-κB-Luciferase activity as following: Clones were harvested inselection medium and counted using a Cell Counter Vi-cell xr 2.03(Beckman Coulter, Cat. No. 731050). Cells were set to a cell density of0.33×10⁶ cells/mL and 150 μL of this cell suspension were transferred toeach well of a sterile white 96-well flat bottom tissue culture platewith lid (greiner bio one, Cat. No. 655083) and, as a control, to aconventional 96 well flat bottom tissue culture plate (TPP Cat. No.92096) to test survival and cell density on the next day. Cells wereincubated at 37° C. and 5% CO₂ overnight. On the next day 50 μL ofmedium containing recombinant human tumor necrosis factor alpha (rhTNFα,PeproTech, Cat.-No. 300 01A) at different concentrations were added toeach well of the white 96-well plate resulting in final concentrationsof 100, 50, 25, 12.5, 6.25 and 0 ng/well rhTNFα. Cells were incubatedfor 6 hours at 37° C. and 5% CO₂ and washed afterwards three times with200 μL/well DPBS. 30 μl freshly prepared Reporter Lysis Buffer (Promega,Cat-No: E3971) were added to each well and the plates were stored overnight at −20° C. The next day frozen cell plates and detection buffer(Luciferase 1000 Assay System, Promega, Cat. No. E4550) were thawed toroom temperature. 100 μL of detection buffer were added to each well andthe plate was measured immediately using the SpectraMax M5/M5emicroplate reader and SoftMax Pro Software (Molecular Devices) withfollowing settings: for luciferase (RLUs), 500 ms integration time, nofilter, collecting all wave length and top reading. Measured URLs abovecontrol (no rhTNFa added) were taken as luciferase activity. TheNF-κc-4-1BB-HeLa clone 26 was chosen for further use exhibiting thehighest luciferase activity and a sensible 4-1BB-expression.

11.1.2. NFKB activation in HeLa cells expressing human 4-1BB co-culturedwith crosslinking Anti-Human IgG1 Fcγ-Fragment Specific Goat IgG F(ab′)₂Fragment

NF-κB-luciferase Human-4-1BB HeLa Cells were Harvested and Resuspendedin DMEM Medium supplied with 10% Fetal Bovine Serum and 1% GlutaMAX Itoa concentration of 0.8×10⁶ cells/mL. 100 μL (8×104 cells) of this cellsuspension were transferred to each well of a sterile white 96-well flatbottom tissue culture plate with lid (greiner bio one, Cat. No. 655083)and the plate were incubated at 37° C. and 5% CO₂ overnight. The nextday 50 μL/well of medium containing titrated FAP- or DP47-targetedsingle chain trimeric human 4-1BBL antigen binding molecules were added.For crosslinking 50 μL/well of medium containing secondary antibodyanti-human IgG Fcγ-fragment-specific goat IgG F(ab′)₂ fragment (JacksonImmunoResearch, Cat. No. 109-006-098) were added in a 1:4 ratio (4 timesmore secondary antibody than the primary single chain trimeric human4-1BBL antigen binding molecule).

Plates were incubated for 6 hours at 37° C. and 5% CO₂. Luciferaseactivity was measured as described under Example 11.1.1. The principleof the assay is illustrated in FIG. 15.

As shown in FIG. 16, crosslinking via a secondary polyclonal anti-huIgG1Fcγ-specific goat IgG F(ab)₂ fragment of FAP-targeted single chaintrimeric human 4-1BBL (closed circle) or DP47-containing single chaintrimeric human 4-1BBL (open circle, dotted line) leads to similaractivation of the NFκB promoter as assessed by the level of luciferaseexpression in the reporter cell line. Therefore, following theirartificially induced non-specific oligomerization, the DP47-cotainingand FAP-targeted single chain trimeric human 4-1BBL molecules showcomparable capacity to agonistically engage 4-1BB at the cell surface.

11.1.3. NFκB Activation in HeLa Cells Expressing Human 4-1BB withFAP-Expressing Tumor Cells

NFκB-luciferase human-4-1BB HeLa cells were harvested and resuspended inDMEM

Medium supplied with 10% FBS and 1% GlutaMAX Ito a concentration of0.2×10⁶ cells/mL. 100 μL (2×10⁴ cells) of this cell suspension weretransferred to each well of a sterile white 96-well flat bottom tissueculture plate with lid (greiner bio one, Cat. No. 655083) and the platewere incubated at 37° C. and 5% CO₂ overnight. On the next day 50 μL ofmedium containing titrated FAP- or DP47-targeted single chain trimerichuman 4-1BBL antigen binding molecule were added.

The following human FAP-expressing tumor cells were resuspended in DMEMsupplied with 10% FBS and 1% GlutaMAX-I to a concentration of 2×10⁶cells/mL:

-   -   human melanoma cell line MV3 (first published in van Muijen GN        et al. 1991),    -   human female melanoma WM-266-4 cell line (ATCC No. CRL-1676),        and    -   mouse embryonic fibroblast NIH/3T3 (ATCC No. CRL-1658)        transfected with expression vector pETR4921 to express human FAP        and Puromycin resistance: NIH/3T3-huFAP clone 39 as described in        Example 10.2.

50 μL of FAP-expressing tumor cell suspension were added to each well.After adding crosslinking FAP-expressing tumor cells or medium (nocrosslinking), plates were incubated for 6 hours at 37° C. and 5% CO₂.Luciferase activity was measured as described in Example 11.1.1.Incubation time and ratio between NFKB-Luciferase human-CD137 HeLa cellsand 3T3-human FAP clone 39 cells were titrated and time and ratio wereset to values reaching the luciferase activation plateau. The principleof the assay is shown in FIG. 17.

As shown in FIGS. 18A to 18D, FAP-targeted single chain 4-1BBLtrimer-containing Fc (kih) fusion antigen binding molecules (CompoundSi) (filled circles) can be crosslinked via FAP-expressing tumor cellsthat leads to NFκB-mediated luciferase-activation in the reporter cellline in a concentration-dependent manner. In contrast, theDP47-containing single chain trimeric human 4-1BBL (Control 1) is unableto activate the reporter cell line under the same experimentalconditions. In the absence of crosslinking (no addition ofFAP-expressing tumor cells) both molecules are unable to induceNFκB-mediated luciferase-activation in the reporter cell line.

As shown in FIGS. 19A to 19C, monovalent or bivalent FAP (4B9 or28H1)-targeted single chain trimeric 4-1BBL Fc (kih) fusion antigenbinding molecules can be crosslinked via FAP-expressing tumor cells andtherefore can induce NF-κB-mediated luciferase-activation in thereporter cell line HeLa-hu4-1BB-NF-κB-luc in a concentration-dependentmanner. In contrast, monovalent or bivalent DP47-untargeted single chaintrimeric 4-1BBL Fc (kih) fusion molecules (Controls 1 and 2) are unableto activate the reporter cell line without cross-linking. In the absenceof crosslinking (no addition of FAP-expressing tumor cells) allmolecules are unable to induce NFκB-mediated luciferase-activation inthe reporter cell line. The monovalent FAP-targeted single chaintrimeric 4-1BBL Fc(kih) fusion antigen binding molecules induce astronger activation than the bivalent FAP (4B9)-targeted single chaintrimeric 4-1BB ligand (kih) fusion molecule, which reflects also thebinding to FAP-expressing tumor cells. The differences between the 4B9and the 28H1 FAP-binding clones however are not as strong as in thebinding assay to FAP-expressing tumor cells. Still the monovalent FAP(4B9)-targeted single chain trimeric 4-1BB ligand (kih) fusion moleculeshows a slightly lower EC₅₀ value which can be explained by the higheraffinity to FAP.

11.1.4. NFκB Activation in HeLa Cells Expressing Human 4-1BB withCEA-Expressing Tumor Xells

The NF-κB activation assay using HeLa-human 4-1BB-NF-κB-luc reportercell together with CEA-expressing tumor cell lines has been performed inthe same way as described in 11.1.3., however the FAP-expressing tumorcells were exchanged with CEA-expressing MKN45 tumor cell line describedalready in Example 10.3 in this application.

As shown in FIGS. 20A and 20B, monovalent and bivalent CEA(sm9b)-targeted single chain trimeric 4-1BBL Fc(kih) fusion antigenbinding molecules can be crosslinked via CEA-expressing tumor cells andtherefore can induce NF-κB-mediated luciferase-activation in thereporter cell line HeLa-hu4-1BB-NF-κB-luc in a concentration-dependentmanner. In contrast monovalent or bivalent DP47-untargeted single chaintrimeric 4-1BBL Fc(kih) fusion molecules (Controls 1 and 2) are unableto activate the reporter cell line without cross-linking. In the absenceof crosslinking (no addition of CEA-expressing tumor cell line MKN45)all molecules are unable to induce NFKB-mediated luciferase-activationin the reporter cell line. The monovalent CEA (sm9b)-targeted singlechain trimeric 4-1BB ligand (kih) fusion molecules (Compound S5) inducea stronger activation than the bivalent CEA (sm9b)-targeted single chaintrimeric 4-1BB ligand (kih) fusion molecules (Compound S4), whichreflects also the MFI of binding to CEA-expressing human MKN45 tumorcell line shown in FIG. 13E.

11.1.5. HeLa Cells Expressing Human OX40 and Reporter GeneNFκB-luciferase

Agonstic binding of OX40 to its ligand induces downstream signaling viaactivation of NFκB (Weinberg et al., 2004). The recombinant reportercell line HeLa_hOx40_NFκB_Luc1 was generated to express human OX40 onits surface. Additionally, it harbors a reporter plasmid containing theluciferase gene under the control of an NFκB-sensitive enhancer region.Ox40 triggering induces dose-dependent activation of NFκB, whichtranslocates in the nucleus, where it binds on the NFκB sensitiveenhancer of the reporter plasmid to increase expression of theluciferase protein. Luciferase catalyzes luciferin-oxidation resultingin oxyluciferin which emits light. This can be quantified by aluminometer. The scope of one experiment was to test the capacity of thesingle chain trimeric human OX40L antigen binding molecule to induceNFκB activation in HeLa_hOx40_NFκB_Luc1 cells.

Morris et al. showed that agonistic co-stimulation with conventionalanti-Ox40 antibodies relied on surface immobilization (Morris et al.,2007). Thus, to achieve a higher degree of hypercrosslinking of OX40,FAP-targeted single chain trimeric human OX40L antigen binding moleculeswere hyper-crosslinked with WM-266-4 tumor cells naturally expressingFAP.

11.1.6. NFκB Activation in HeLa Cells Expressing Human OX40 withFAP-Expressing Tumor Cells

All centrifugations were done at 350×g for 5 minutes at 4° C. To washthe cells, the tubes or wells were filled up with the indicated buffer.After centrifugation the supernatant was aspirated and the cell pelletresuspended in the indicated buffer.

Adherent HeLa hOX40_NFκB_Luc1 cells and WM-266-4 cells were harvestedusing cell dissociation buffer (Invitrogen, Cat.-No. 13151-014) for 10minutes at 37° C. Cells were washed once with DPBS. HeLa_hOX40_NFκB_Luc1cells were adjusted to a cell density of 2×10⁵ cells/mL in assay mediumcontaining MEM (Invitrogen, Cat.-No. 22561-021), 10% (v/v)heat-inactivated FBS, 1 mM sodium pyruvate and 1% (v/v) non-essentialamino acids. Cells were seeded in a density of 0.3×10⁵ cells per well ina sterile white 96-well flat bottom tissue culture plate with lid(greiner bio one, Cat. No. 655083) and kept over night at 37° C. and 5%CO₂ in an incubator (Hera Cell 150). On the next day,HeLa_hOX40_NFκB_Luc1 were stimulated for 6 hours adding assay mediumcontaining titrated FAP-targeted or DP47-containing single chaintrimeric human OX40L antigen binding molecules. For hyper-crosslinkinghuman FAP-expressing WM266-4 tumor cells were adjusted to a cell densityof 3×106 cells/mL in assay media and cells were added to a final densityof 0.75×10⁵ cells/well.

After 6 hours of incubation at 37° C. and 5% CO₂ in an incubator,supernatant was aspirated and plates washed two times with DPBS.Quantification of light emission was done using the luciferase 100 assaysystem and the reporter lysis buffer (both Promega, Cat. No. E4550 andCat-No: E3971) according to manufacturer instructions.

Emitted relative light units (URL) were corrected by basal luminescenceof HeLa_hOX40_NFκB_Luc1 cells and were blotted against the logarithmicprimary antibody concentration using Prism4 (GraphPad Software, USA).Curves were fitted using the inbuilt sigmoidal dose response.

As shown in FIGS. 21A-B, both the FAP-targeted and DP47-containingsingle chain trimeric human OX40L Fc(kih) fusion molecules (filled andopen circles) induced detectable NFKB activation. This is in contrast towhat was observed with the single chain trimeric human 4-1BBL moleculeswhich showed no NFkB-inducing activity without hyper-crosslinkingthrough secondary antibodies. Hyper-crosslinking via FAP-expressingtumor cell WM-266-4 strongly increased induction of NFκB-mediatedluciferase expression in a concentration-dependent manner by theFAP-targeted single chain trimeric human OX40L Fc(kih) fusion antigenbinding molecule (Compounds S10, filled circle). No such effect was seenwhen the DP47-containing single chain trimeric human OX40L Fc(kih)fusion molecule was used.

11.2 Antigen-Specific CD8+ T Cell-Based Assay

11.2.1. Isolation and Culture of Antigen-Specific CD8 T Cells

Fresh blood was obtained from a HLA-A2+ CMV-infected volunteer. PBMCswere isolated as described in Example 10.1 by ficoll densitycentrifugation. CD8 T cells were purified from PBMCs using a negativeselection human CD8 T cell isolation kit according to manufacturer'srecommendations (Miltenyi Biotec, Cat. No. 130-094-156). Ten million ofisolated CD8 T cells were resuspended in 1 mL sterile DPBS supplementedwith 1% (v/v) FBS along with 50 μL of PE-labeled HLA-A2-pentamercontaining the CMV-derived NLVPMVATV peptide (ProImmune, Cat. No.F008-2B, SEQ ID NO:183) and incubated for 10 min at room temperature.Cells were washed twice with 3 mL sterile DPBS supplied with 1% (v/v)FBS. Cells were resuspended in 1 mL DPBS supplied with 1% (v/v) FBScontaining 1 μg/mL anti-human CD8-FITC (clone LT8, Abcam, Cat. No.Ab28010) and incubated for 30 minutes at 4° C. Then, the cells werewashed twice, resuspended to a concentration of 5×10⁶ cells/mL in DPBSsupplied with 1% (v/v) FBS, and filtrated through a 30 μm pre-separationnylon-net cell strainer (Miltenyi Biotec, Cat. No. 130-041-407).NLV-peptide-specific CD8⁺ T cells were isolated by FACS sorting using anARIA cell sorter (BD Bioscience with DIVA software) with the followingsettings: 100 μm nozzle and purity sort mask. Sorted cells werecollected in a 15 ml polypropylene centrifuge tube (TPP, Cat. No. 91015)containing 5 ml RPMI 1640 medium supplied with 10% (v/v) FBS, 1% (v/v)GlutaMAX-I and 400 U/mL Proleukin. Sorted cells were centrifuged for 7minutes at 350×g at room temperature and resuspended in same medium to aconcentration of 0.53×10⁶ cells/mL. 100 μL/well of this cell suspensionwere added to each well of a previously prepared plate withPHA-L-activated irradiated allogeneic feeder cells. Feeder cells wereprepared from PBMCs as previously described (Levitsky et al., 1998) anddistributed to 96-well culture plates using 2×10⁵ feeder cells per well.

After one day of culture, 100 μL medium were removed from each well andreplaced by new RPMI 1640 medium supplemented with 10% (v/v) FBS and 1%(v/v) GlutaMAX-I and 400 U/mL Proleukin. This was repeated duringculture on a regular basis (every 2-4 days). As soon as NLV-specificCD8⁺ T cells started to proliferate, they were transferred to 24-wellflat-bottom tissue culture plate (TPP, 92024). Cells were expanded/splitand reactivated with new feeder cell preparation on a regular basis.

11.2.2. Activation Assay of Antigen-Specific CD8⁺ T Cells

Cells of the human FAP-expressing melanoma cell line MV3 cell line(described in Example 11.1.3.) were harvested and washed with DPBS and2×10⁷ cells were resuspended in 250 μL C diluent of the PKH-26 RedFluorescence Cell linker Kit (Sigma, Cat.-No. PKH26GL). 1μLPKH26-Red-stain solution was diluted with 250 μL C diluent and added tothe suspension of MV3 cells which were then incubated for 5 min at roomtemperature in the dark. 0.5 mL FBS were added, cells were incubated for1 minute and washed once with T cell medium consisting of RPMI 1640medium supplemented with 10% (v/v) FBS, 1% (v/v) GlutaMAX-I, 1 mM sodiumpyruvate, 1% (v/v) MEM non-essential amino acids and 50 μMβ-Mercaptoethanol. 1×106 MV3 cells/mL were resuspended in T cell mediumand separated into three tubes. Synthetic NLVPMVATV peptide (obtainedfrom thinkpeptides, SEQ ID NO: 181) was added to a final concentrationof 1×10⁻⁹M or 1×10⁻⁸M and cells were incubated for 90 min under rotationat 37° C. and 5% CO₂. MV3 cells were washed once with T cell medium andresuspended to a density of 0.5×10⁶ cells/mL, distributed (100 μL/well)to a 96-well round bottom cell-suspension plate (Greiner bio-one,cellstar, Cat. No. 650185) and incubated over night at 37° C. and 5%CO₂.

On the next day, FAP-targeted and DP47-containing single chain trimerichuman 4-1BBL molecules were added with 50 μL of T-cell medium to achievethe indicated final concentrations.NLV-specific CD8 T cells wereharvested, washed and added in 50 μL medium to each well (final tumor:CD8 T cell ratio 0.125). Cells were incubated for 24 h and 50 μL/well Tcell medium containing 2.64 μL/mL Golgi stop (Protein TransportInhibitor containing Monesin, BD Bioscience, Cat.-No. 554724) were addedto each well. Cells were incubated for additional 4 h, washed with 200μL/well DPBS and stained with 100 μL DPBS containing 1:5000 dilutedFixable Viability Dye-eF450 (eBioscience, Cat. No. 65-0864) for 30minutes at 4° C. Cells were washed with DPBS and stained in 40 μL/wellFACS buffer containing the following fluorescent dye-conjugatedantibodies: anti-human CD137-PerCP/Cy5.5 (clone 4B4-1, mouse IgG1κ,BioLegend, Cat. No. 309814), anti-human CD8-BV605 (clone RPA T8, mouseIgG1κ, BioLegend, Cat. No. 301012) and anti-human CD25 PE/Cy7 (cloneBC96, mouse IgG1κ, BioLegend, Cat. No. 302612). After incubation for 30min at 4° C., cells were washed twice with 200 μL/well FACS buffer,resuspended in 50 μL/well freshly prepared FoxP3 Fix/Perm buffer(eBioscience Cat.-No. 00-5123 and 00-5223) and incubated for 30 min at4° C. Plates were washed twice with 200 μL/well Perm-Buffer (DPBSsupplied with 2% (v/v) FBS, 1% (w/v) saponin (Sigma Life Science, 57900)and 1% (w/v) sodium azide (Sigma-Aldrich, 52002) and stained with 50μL/well Perm-Buffer (eBioscience, Cat.-No. 00-8333-56) containing 0.25μg/mL anti-human IFNγ-APC (clone B27, mouse IgG1κ, BioLegend, Cat. No.506510). Plates were incubated for 1 h at 4° C. and washed twice with200 μL/well Perm-Buffer. For fixation, 50 μL/well DPBS containing 1%formaldehyde were added and cells were stored overnight at 4° C. Thenext day, cells were resuspended in 100 μL/well FACS buffer and acquiredusing a 5-laser Fortessa flow cytometer (BD Bioscience with DIVAsoftware). The principle of the assay is shown in FIG. 22.

As shown in FIGS. 23A to 23F, antigen-specific stimulated CD8⁺ T cells,but not unstimulated controls, exhibited increased levels of surface4-1BB expression in the presence of FAP-targeted single chain trimerichuman 4-1BBL Fc(kih) fusion antigen binding molecule (Compound 51,filled circles). This effect of trimeric human 4-1BBL was dose dependentand required FAP-targeting as addition of the untargeted controlmolecule (Control 1) did not affect the level of 4-1BB expression.Furthermore, T cells activated at a higher peptide concentration (1×10⁻⁸M) showed sustained secretion of INFγ in the presence of FAP-targetedsingle chain trimeric human 4-1BBL (FIGS. 24A to 24F). Notably, thiseffect required a stronger primary TCR triggering as compared to theeffect of the FAP-targeted single chain trimeric human 4-1BBL on 4-1BBupregulation as the latter was clearly observed also at a lowconcentration of the peptide. Collectively, these data demonstrate thatthe FAP-targeted single chain trimeric human 4-1BBL Fc(kih) fusionantigen binding molecule modulates the surface phenotype andresponsiveness of antigen specific T-cells in a targeting dependentmanner.

As shown in FIGS. 25A-F and 26A-F, antigen-specific CD8+ T cellsco-stimulated with NLV-peptide (activation signal 1) and crosslinkedmonovalent or bivalent FAP (28H1 or 4B9)-targeted single chain trimeric4-1BBL Fc(kih) fusion antigen binding molecules (activation signal 2),but not unstimulated controls, exhibited increased levels of surface4-1BB expression and IFNγ-secretion. This effect was dose dependent forthe NLV-peptide concentration as well as for the concentration ofFAP-targeted single chain trimeric 4-1BBL Fc(kih) fusion antigen bindingmolecule. Collectively, these data demonstrate that the FAP-targetedsingle chain trimeric 4-1BBL Fc(kih) fusion antigen binding moleculesmodulate the surface phenotype and responsiveness of antigen specificT-cells in a targeting dependent manner. The same difference between themonovalent FAP (28H1)-targeted and the FAP (4B9)-targeted single chaintrimeric 4-1BB ligand (kih) fusion molecules are seen as in theactivation assay using the HeLa-hu4-1BB-NK-κB-luc reporter cell line,e.g. the monovalent FAP (4B9)-targeted single chain trimeric 4-1BBLFc(kih) fusion antigen binding molecule shows lower EC₅₀ values.Different to the HeLa-hu 4-1BB-NK-κB-luc reporter cell line activationassay no big differences are seen between the monovalent and thebivalent FAP (4B9)-targeted single chain trimeric 4-1BB ligand (kih)fusion molecules. At high NLV-peptide concentrations (10⁻⁸M) it evensseems, that the bivalent FAP (4B9)-targeted single chain trimeric 4-1BBLFc(kih) fusion antigen binding molecule produces a superior activationthan the monovalent FAP (4B9)-targeted single chain trimeric 4-1BBLFc(kih) fusion antigen binding molecule.

11. 3. OX40 Mediated Costimulation of Suboptimally TCR Triggered RestingHuman PBMC

Human PBMC preparations contain (1) resting OX40 negative CD4⁺ and CD8⁺T cells and (2) antigen presenting cells with various Fc-y receptormolecules on their cell surface, e.g. B cells and monocytes. Anti-humanCD3 antibody of human IgG1 isotype can bind with its Fc part to Fc-yreceptor molecules and mediate TCR triggering on resting OX40 negativeCD4⁺ and CD8⁺ T cells which start to express OX40 within several hours.Functional agonistic compounds against OX40 can signal via the OX40receptor present on activated CD8⁺ and CD4⁺ T cells and supportTCR-mediated stimulation, leading to more prominent phenotypic changes(e.g. higher CD25 expression levels), enhanced proliferation andincreased numbers of living CD4⁺ and CD8⁺ T cells.

Experiments with HeLa_hOx40_NFκB_Luc1 reporter cell lines suggested acertain intrinsic capacity of trimeric single chain Ox40L molecules toinduce NFKB expression (see Example 11.1.5.). The agonistic effect wasgreatly increased by additional hypercrosslinking of the FAP-targetedcompound via FAP expressing tumor cells. Thus, suboptimal TCRstimulation of resting human PBMC was performed in the presence ofNIH/3T3-huFAP clone 39 cells and DP47-containing (untargeted) andFAP-targeted trimeric single chain Ox40L molecules to test thecostimulatory activity with and without further hypercrosslinking.

Mouse embryonic fibroblast NIH/3T3-huFAP clone 39 cells (see Example9.2.) were harvested using cell dissociation buffer (Invitrogen,Cat.-No. 13151-014) for 10 minutes at 37° C. Cells were washed once withDPBS. NIH/3T3-huFAP clone 39 cells were adjusted to a cell density of1.6×10⁵ in T cell media and were seeded at a density of 0.2×10⁵ cellsper well in a sterile 96-well round bottom adhesion tissue culture plate(TPP, Cat. No. 92097). Plates were kept overnight at 37° C. and 5% CO₂in an incubator (Hera Cell 150) before they were irradiated the next dayin an xRay irradiator using a dose of 4500 RAD to prevent laterovergrowth of human PBMC by the cell line.

Human PBMCs were isolated as described under 9.1. by ficoll densitycentrifugation and were labeled with CFSE. Cells were stained at a celldensity of 1×10⁶ cells/ mL with CFDA-SE (Sigma-Aldrich, Cat.-No. 2188)at a final concentration of [50 nM] for 10 minutes at 37° C. Thereafter,cells were washed twice with excess DPBS containing FBS (10% v/v).Labeled cells were rested in T cell media at 37° C. for 30 minutes.Thereafter, non-converted CFDA-SE was removed by two additional washingsteps with DPBS. CFSE-labeled resting human PBMC were adjusted to a celldensity of 3×10⁶ cells/mL in T cell media and were added to each well ata density of 0.75×10⁵ cells per well. FAP-targeted and DP47-containingsingle chain trimeric Ox40L antigen binding molecules were added at theindicated concentrations and anti-human CD3 antibody (clone V9, humanIgG1) at a final concentration of 10 nM. Plates were kept for four daysat 37° C. and 5% CO₂. After 48 hours a half medium exchange wasperformed.

After four days cells were washed with DPBS and then stained in 25μL/well FACS buffer containing following fluorescent dye-conjugatedantibodies: 0.250 μg/mL anti-human CD4-BV421 (clone RPA-T4, mouse IgG1k, BioLegend, Cat.-No. 300532), 0.250 μg/mL CD8-BV711 (clone RPa-T8,mouse IgG1k, BioLegend, Cat.-No. 3010441, 0.250 μg/mL anti-humanCD25-PerCP Cy5.5 (clone M-A251, mouse IgGlk, BioLegend, Cat.-No.356112). After incubation for 30 min at 4° C., cells were washed twicewith 200 μL/well FACS buffer, resuspended in 50 μL/well freshly preparedFoxP3 Fix/Perm buffer (eBioscience Cat.-No. 00-5123 and 00-5223) andincubated for 45 min at room temperature in the dark. Plates were washedthree times with 200 μL/well Perm-Wash-(eBioscience, Cat.-No.00-8333-56) and were then stained with 25 μL/well Perm-Buffer(eBioscience, Cat.-No. 00-8333-56) containing 0.25 μg/mL anti-humanGranzyme B—PE (clone GB-11, mouse IgG1κ, BD Bioscience, Cat. No. 561142)and 0.60 μg/mL anti-human Eomes-eFluor 670 (clone WD2918, mouse IgG1κ,eBioscience, Cat. No. 50-4877-42). Plates were incubated for 1 h at roomtemperature in the dark and were washed twice with 200 μL/wellPerm-Buffer. Cells were resuspended in 85 μL/well FACS buffer andacquired using a 5-laser Fortessa flow cytometer (BD Bioscience withDIVA software).

As shown in FIGS. 27A-F, costimulation with DP47-containing trimericsingle chain Ox40L antigen binding molecules only slightly promotedproliferation and enhanced the activated cell phenotype (CD25expression) in human CD4⁺ (left side) and CD8⁺ T cells (right side)stimulated sub-optimally with anti-human CD3 antibody.Hyper-crosslinking of the FAP-targeted trimeric single chain Ox40L byNIH/3T3-huFAP clone 39 cells strongly increased this effect. This is incontrast to findings in the HeLa_hOx40_NFκB_Luc1 cells (FIGS. 21A-B),where the trimeric single chain Ox40L molecule per se was alreadystrongly agonistic. This might be due to a non-physiologically highexpression of OX40 in the reporter cell line, which most likely resultsin pre-assembly of low-signaling OX4OR oligomer units thereby loweringthe threshold for agonistic signaling is the reporter cell line ascompared to that Ox40⁺ T cells. In addition, the cellular context islikely to determine the outcome of OX40 triggering as concomitantsignaling through TCR is required in T-cells, but not in the reportercell line, to observe the effect of OX40 engagement.

CITATIONS

Aggarwal B. B. (2003), Signalling pathways of the TNF superfamily: adouble-edged sword. Nat. Rev. Immunol. 3(9),745-56.

Banner D. et al (1993), Crystal structure of the soluble human 55 kd TNFreceptor-human TNF beta complex: implications for TNF receptoractivation. Cell 73, 431-445.

Baumann R., Shida Y., Simon D., Russmann S., Mueller C. and Simon H.-U.(2004), Functional expression of CD134 by neutrophils. Eur. J. Immunol.,34, 2268-2275.

Bodmer J., Schneider P. and Tschopp, J. (2002), The moleculararchitecture of the TNF superfamily. Trends in Biochemical Sciences27(1), 19-26.

Bremer E. (2013), Targeting of the tumor necrosis factor receptorsuperfamily for cancer immunotherapy. 2013, Article ID 371854, 25 pages.doi:10.1155/2013/371854

Broll K., Richter G., Pauly S., Hofstaedter F. and Schwarz, H. (2001),CD137 expression in tumor vessel walls. High correlation with malignanttumors. Am J Clin Pathol 115, 543-549.

Buechele C., Baessler T., Schmiedel B. J., Schumacher C. E.,Grosse-Hovest L., Rittig K. and Salih, H .R. (2012). 4-1BB ligandmodulates direct and Rituximab-induced NK-cell reactivity in chroniclymphocytic leukemia. Eur J Immunol 42, 737-748.

Carter P. (2001). Bispecific human IgG by design. J. Immunol. Methods248, 7-15.

Choi B.K., Kim Y. H., Kwon P. M., Lee S. C., Kang S. W., Kim M. S., LeeM. J., and Kwon B. S. (2009). 4-1BB functions as a survival factor indendritic cells. J Immunol 182, 4107-4115.

Croft M., Song T., Duan W. and Soroash P. (2009). The significance of0X40 and OX40L to T-cell biology and immune disease. ImmunologicalReviews 229, 173-191.

Cuadros C., Dominguez A. L., Lollini P. L., Croft M., Mittler R. S.,Borgstrom P., and Lustgarten J. (2005). Vaccination with dendritic cellspulsed with apoptotic tumors in combination with anti-OX40 andanti-4-1BB monoclonal antibodies induces T cell-mediated protectiveimmunity in Her-2/neu transgenic mice. Int J Cancer 116, 934-943.

Curran M. A., Kim M., Montalvo W., Al-Shamkhani A., and Allison J. P.(2011). Combination CTLA-4 blockade and 4-1BB activation enhances tumorrejection by increasing T-cell infiltration, proliferation, and cytokineproduction. PLoS One 6, e19499.

Diehl L., van Mierlo G. J., den Boer A. T., van der Voort E., FransenM., van Bostelen L., Krimpenfort P., Melief C. J., Mittler R., Toes R.E., and Offringa R. (2002). In vivo triggering through 4-1BB enablesTh-independent priming of CTL in the presence of an intact CD28costimulatory pathway. J Immunol 168, 3755-3762.

Dubrot J., Milheiro F., Alfaro C., Palazon A., Martinez-Forero I.,Perez-Gracia J. L Morales-Morales-Kastresana A., Romero-Trevejo J.L.,Ochoa M.C., Hervas-Stubbs S., et al. (2010). Treatment with anti-CD137mAbs causes intense accumulations of liver T cells without selectiveantitumor immunotherapeutic effects in this organ. Cancer ImmunolImmunother 59, 1223-1233.

Futagawa T., Akiba H., Kodama T., Takeda K., Hosoda Y., Yagita H., andOkumura K. (2002). Expression and function of 4-1BB and 4-1BB ligand onmurine dendritic cells. Int Immunol 14, 275-286.

Graff, C. P., Chester K., Begent R. and Wittrup K. D. (2004). Directedevolution of an anti-carcinoembryonic antigen scFv with a 4-daymonovalent dissociation half-time at 37° C. Protein Engineering, Design& Selection 17, 293-304.

Guo Z., Cheng D., Xia Z., Luan M., Wu L., Wang G., and Zhang S. (2013).Combined TIM-3 blockade and CD137 activation affords the long-termprotection in a murine model of ovarian cancer. J Transl Med 11, 215.

Heinisch I. V., Daigle I., Knopfli B., and Simon H. U. (2000). CD137activation abrogates granulocyte-macrophage colony-stimulatingfactor-mediated anti-apoptosis in neutrophils. Eur J Immunol 30,3441-3446.

Hornig, N., Kermer, V., Frey, K., Diebolder, P., Kontermann, R. E.,Mueller, D. (2012), Combination of a bispecific antibody andcostimulatory antibody-ligand fusion proteins for targeted cancerimmunotherapy. J. Immunother. 35, 418-429.

Ju S. A., Cheon S. H., Park S. M., Tam N. Q., Kim Y. M., An W. G., andKim B. S. (2008). Eradication of established renal cell carcinoma by acombination of 5-fluorouracil and anti-4-1BB monoclonal antibody inmice. Int J Cancer 122, 2784-2790.

Kermer V., Hornig N., Harder M., Bondarieva A., Kontermann R.E., andMuller D. (2014). Combining Antibody-directed presentation of IL-15 and4-1BBL in a trifunctional fusion protein for Cancer Immunotherapy. Mol.Cancer Ther. 13, 112-121.

Kienzle G., and von Kempis J. (2000). CD137 (ILA/4-1BB), expressed byprimary human monocytes, induces monocyte activation and apoptosis of Blymphocytes. Int Immunol 12, 73-82.

Kim D. H., Chang W. S., Lee Y. S., Lee K. A., Kim Y. K., Kwon B. S., andKang C. Y. (2008). 4-1BB engagement costimulates NKT cell activation andexacerbates NKT cell ligand-induced airway hyperresponsiveness andinflammation. J Immunol 180, 2062-2068.

Kim Y. H., Choi B. K., Oh H. S., Kang W. J., Mittler R. S., and Kwon B.S. (2009). Mechanisms involved in synergistic anticancer effects ofanti-4-1BB and cyclophosphamide therapy. Mol Cancer Ther 8, 469-478.

Kwon B. S., and Weissman S. M. (1989). cDNA sequences of two inducibleT-cell genes. Proc Natl Acad Sci USA 86, 1963-1967.

Lee H., Park H. J., Sohn H. J., Kim J. M., and Kim S. J. (2011).Combinatorial therapy for liver metastatic colon cancer: dendritic cellvaccine and low-dose agonistic anti-4-1BB antibody co-stimulatorysignal. J Surg Res 169, e43-50.

Levitsky V., de Campos-Lima P. O., Frisan T., and Masucci M. G. (1998).The clonal composition of a peptide-specific oligoclonal CTL repertoireselected in response to persistent EBV infection is stable over time. JImmunol 161, 594-601.

Li F., and Ravetch J. V. (2011). Inhibitory Fcgamma receptor engagementdrives adjuvant and anti-tumor activities of agonistic CD40 antibodies.Science 333, 1030-1034.

Lin W., Voskens C. J., Zhang X., Schindler D. G., Wood A., Burch E., WeiY., Chen L., Tian G., Tamada K., et al. (2008). Fc-dependent expressionof CD137 on human NK cells: insights into “agonistic” effects ofanti-CD137 monoclonal antibodies. Blood 112, 699-707.

Melero I., Johnston J. V., Shufford W. W., Mittler R. S., and Chen L.(1998). NK1.1 cells express 4-1BB (CDw137) costimulatory molecule andare required for tumor immunity elicited by anti-4-1BB monoclonalantibodies. Cell Immunol 190, 167-172.

Melero I., Shuford W. W., Newby S. A., Aruffo A., Ledbetter J. A.,Hellstrom K. E., Mittler R. S., and Chen L. (1997). Monoclonalantibodies against the 4-1BB T-cell activation molecule eradicateestablished tumors. Nat Med 3, 682-685.

Merchant A. M., Zhu Z., Yuan J. Q., Goddard A., Adams C. W., Presta L.G., and Carter P. (1998). An efficient route to human bispecific IgG.Nat Biotechnol 16, 677-681.

Morales-Kastresana A., Sanmamed M. F., Rodriguez I., Palazon A.,Martinez-Forero I., Labiano S., Hervas-Stubbs S., Sangro B., Ochoa C.,Rouzaut A., et al. (2013). Combined immunostimulatory monoclonalantibodies extend survival in an aggressive transgenic hepatocellularcarcinoma mouse model. Clin Cancer Res 19, 6151-6162.

Morris N. P., Peters C., Montler R., Hu H-M, Curti B. D., Urba W. J.,and Weinberg A. D. (2007). Development and Characterization ofRecombinant Human Fc:OX40L fusion protein linked via a coiled-coiltrimerization domain. Mol. Immunol. 44(12), 3112-3121.

Mueller, D., Frey, K., Kontermann, R. E. (2008), A novel antibody-4-1BB1fusion protein for targeted costimulation in cancer immunotherapy, J.Immunother. 31, 714-722.

Murillo O., Dubrot J., Palazon A., Anna A., Azpilikueta A., Alfaro C.,Solano S., Ochoa M. C., Berasain C., Gabari I., et al. (2009). In vivodepletion of DC impairs the anti-tumor effect of agonistic anti-CD137mAb. Eur J Immunol 39, 2424-2436.

Narazaki H., Zhu Y., Luo L., Zhu G., and Chen L. (2010). CD137 agonistantibody prevents cancer recurrence: contribution of CD137 on bothhematopoietic and nonhematopoietic cells. Blood 115, 1941-1948.

Nishimoto H., Lee S. W., Hong H., Potter K. G., Maeda-Yamamoto M.,Kinoshita T., Kawakami Y., Mittler R. S., Kwon B .S., Ware C. F., et al.(2005). Costimulation of mast cells by 4-1BB, a member of the tumornecrosis factor receptor superfamily, with the high-affinity IgEreceptor. Blood 106, 4241-4248.

Olofsson P. S., Soderstrom L. A., Wagsater D., Sheikine Y., Ocaya P.,Lang F., Rabu C., Chen L., Rudling M., Aukrust P., et al. (2008). CD137is expressed in human atherosclerosis and promotes development of plaqueinflammation in hypercholesterolemic mice. Circulation 117, 1292-1301.

Palazon A., Teijeira A., Martinez-Forero I., Hervas-Stubbs S., RoncalC., Penuelas I., Dubrot J., Morales-Kastresana A., Perez-Gracia J. L.,Ochoa M. C., et al. (2011). Agonist anti-CD137 mAb act on tumorendothelial cells to enhance recruitment of activated T lymphocytes.Cancer Res 71, 801-811.

Schwarz H., Valbracht J., Tuckwell J., von Kempis J., and Lotz M.(1995). ILA, the human 4-1BB homologue, is inducible in lymphoid andother cell lineages. Blood 85, 1043-1052.

Shao, Z., and Schwarz, H. (2011). CD137 ligand, a member of the tumornecrosis factor family, regulates immune responses via reverse signaltransduction. J Leukoc Biol 89, 21-29.

Shi W., and Siemann D. W. (2006). Augmented antitumor effects ofradiation therapy by 4-1BB antibody (BMS-469492) treatment. AnticancerRes 26, 3445-3453.

Simeone E., and Ascierto P. A. (2012). Immunomodulating antibodies inthe treatment of metastatic melanoma: the experience with anti-CTLA-4,anti-CD137, and anti-PD1. J Immunotoxicol 9, 241-247.

Snell L. M., Lin G. H., McPherson A. J., Moraes T. J., and Watts T. H.(2011). T-cell intrinsic effects of GITR and 4-1BB during viralinfection and cancer immunotherapy. Immunol Rev 244, 197-217.

Song J., So T. and Croft M. (2008). Activation of NF-KB1 by OX40contributes to antigen-driven T cell expansion and survival. J.Immunology 180(11), 7240-7248.

Stagg J., Loi S., Divisekera U., Ngiow S. F., Duret, H., Yagita H., TengM. W., and Smyth M. J. (2011). Anti-ErbB-2 mAb therapy requires type Iand II interferons and synergizes with anti-PD-1 or anti-CD137 mAbtherapy. Proc Natl Acad Sci USA 108, 7142-7147.

Teng M. W., Sharkey J., McLaughlin N. M., Exley M. A., and Smyth M.J .(2009). CD1d-based combination therapy eradicates established tumors inmice. J Immunol 183, 1911-1920.

van Muij en G N, Jansen K F, Cornelissen I M, Smeets D F, Beck J L andRuiter D J (1991). Establishment and characterization of a humanmelanoma cell line (MV3) which is highly metastatic in nude mice. Int JCancer., 48(1), 85-91.

von Kempis J., Schwarz H., and Lotz M. (1997). Differentiation-dependentand stimulus-specific expression of ILA, the human 4-1BB-homologue, incells of mesenchymal origin. Osteoarthritis Cartilage 5, 394-406.

Watts T. H. (2005). TNF/TNFR family members in costimulation of T cellresponses. Annu. Rev. Immunol. 23, 23-68

Wei H., Zhao L., Li W., Fan K., Qian W., Hou S., Wang H., Dai M.,Hellstrom I., Hellstrom K. E., and Guo Y. (2013). Combinatorial PD-1blockade and CD137 activation has therapeutic efficacy in murine cancermodels and synergizes with cisplatin. PLoS One 8, e84927.

Weinberg A D, Evans D E, Thalhofer C, Shi T, Prell R A (2004). Thegeneration of T cell memory: a review describing the molecular andcellular events following OX40 (CD134) engagement. J Leukoc Biol. 75(6),962-972.

Wilcox R. A., Chapoval A. I., Gorski K. S., Otsuji M., Shin T., Flies D.B., Tamada K., Mittler R. S., Tsuchiya H., Pardoll D. M., and Chen L.(2002). Cutting edge: Expression of functional CD137 receptor bydendritic cells. J Immunol 168, 4262-4267.

Wilcox R. A., Tamada K., Flies D. B., Zhu G., Chapoval A. I., Blazar B.R., Kast W. M., and Chen L. (2004). Ligation of CD137 receptor preventsand reverses established anergy of CD8+ cytolytic T lymphocytes in vivo.Blood 103, 177-184.5

Zhang, N., Sadun, R. E., Arias, R. S., Flanagan, M. L., Sachsman, S. M.,Nien, Y, Khawli, L. A., Hu, P., Epstein, A. L. (2007). Targeted anduntargeted CD137L fusion proteins for the immunotherapy of experimentalsolid tumors. Clin. Cancer Res. 13, 2758-2767.

Zhang X., Voskens C. J., Sallin M., Maniar A., Montes C. L., Zhang Y.,Lin W., Li G., Burch E., Tan M., et al. (2010). CD137 promotesproliferation and survival of human B cells. J Immunol 184, 787-795.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1. A TNF family ligand trimer-containing antigen binding moleculecomprising (a) at least one moiety capable of specific binding to atarget cell antigen, (b) a polypeptide comprising three ectodomains of aTNF ligand family member or fragments thereof that are connected to eachother, optionally by peptide linkers, and (c) a Fc domain composed of afirst and a second subunit capable of stable association.
 2. The TNFfamily ligand trimer-containing antigen binding molecule of claim 1comprising (a) at least one moiety capable of specific binding to atarget cell antigen, (b) a polypeptide comprising three ectodomains of aTNF ligand family member or fragments thereof that are connected to eachother by peptide linkers and (c) a Fc domain composed of a first and asecond subunit capable of stable association.
 3. The TNF family ligandtrimer containing antigen binding molecule of claim 1 or 2 comprising(a) at least one moiety capable of specific binding to a target cellantigen, (b) a polypeptide comprising three ectodomains of a TNF ligandfamily member or fragments thereof that are connected to each other bypeptide linkers and (c) a Fc domain composed of a first and a secondsubunit capable of stable association, wherein the polypeptidecomprising the three ectodomains of a TNF ligand family member orfragments thereof that are connected to each other by peptide linkers isfused to the N- or C-terminal amino acid of one of the two subunits ofthe Fc domain, optionally through a peptide linker.
 4. The TNF familyligand trimer-containing antigen binding molecule of any one of claims 1to 3, wherein the TNF ligand family member costimulates human T-cellactivation.
 5. The TNF family ligand trimer-containing antigen bindingmolecule of any one of claims 1 to 4, wherein the TNF ligand familymember is selected from 4-1BBL and OX40L.
 6. The TNF family ligandtrimer-containing antigen binding molecule of any one of claims 1 to 5,wherein the TNF ligand family member is 4-1BBL.
 7. The TNF family ligandtrimer-containing antigen binding molecule of any one of claims 1 to 6,wherein the ectodomain of a TNF ligand family member comprises the aminoacid sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:99, SEQ ID NO: 100, SEQ IDNO:101 and SEQ ID NO:102, particularly the amino acid sequence of SEQ IDNO:1 or SEQ ID NO:99.
 8. The TNF family ligand trimer-containing antigenbinding molecule of any one of claims 1 to 6, comprising (a) at leastone moiety capable of specific binding to a target cell antigen and (b)a polypeptide comprising the amino acid sequence of SEQ ID NO:5 or SEQID NO:103, and (c) a Fc domain composed of a first and a second subunitcapable of stable association.
 9. The TNF family ligandtrimer-containing antigen binding molecule of any one of claims 1 to 5,wherein the TNF ligand family member is OX40L.
 10. The TNF family ligandtrimer-containing antigen binding molecule of any one of claim 1 to 5 or9, wherein the ectodomain of a TNF ligand family member comprises theamino acid sequence selected from the group consisting of SEQ ID NO:6and SEQ ID NO:7, particularly the amino acid sequence of SEQ ID NO:6.11. The TNF family ligand trimer-containing antigen binding molecule ofany one of claim 1 to 5 or 9 or 10, comprising (a) at least one moietycapable of specific binding to a target cell antigen and (b) apolypeptide comprising the amino acid sequence of SEQ ID NO:8, and (c) aFc domain composed of a first and a second subunit capable of stableassociation.
 12. The TNF family ligand trimer-containing antigen bindingmolecule of any one of claims 1 to 11, wherein the polypeptidecomprising three ectodomains of a TNF ligand family member or fragmentsthereof is fused at the C-terminal amino acid to a N-terminal amino acidof one of the subunits of the Fc domain.
 13. The TNF family ligandtrimer-containing antigen binding molecule of any one of claims 1 to 11,wherein the polypeptide comprising three ectodomains of a TNF ligandfamily member or fragments thereof is fused at the N-terminal amino acidto a C-terminal amino acid of one of the subunits of the Fc domain. 14.The TNF family ligand trimer-containing antigen binding molecule of anyone of claims 1 to 13, wherein the moiety capable of specific binding toa target cell antigen is selected from the group consisting of anantibody fragment, a Fab molecule, a crossover Fab molecule, a singlechain Fab molecule, a Fv molecule, a scFv molecule, a single domainantibody, an aVH and a scaffold antigen binding protein.
 15. The TNFfamily ligand trimer-containing antigen binding molecule of any one ofclaims 1 to 14, wherein the moiety capable of specific binding to atarget cell antigen is a Fab molecule capable of specific binding to atarget cell antigen.
 16. The TNF family ligand trimer-containing antigenbinding molecule of any one of claims 1 to 15, wherein the target cellantigen is selected from the group consisting of Fibroblast ActivationProtein (FAP), Melanoma-associated Chondroitin Sulfate Proteoglycan(MCSP), Epidermal Growth Factor Receptor (EGFR), CarcinoembryonicAntigen (CEA), CD19, CD20 and CD33.
 17. The TNF family ligandtrimer-containing antigen binding molecule of any one of claims 1 to 16,wherein the target cell antigen is Fibroblast Activation Protein (FAP).18. The TNF family ligand trimer-containing antigen binding molecule ofany one of claims 1 to 17, wherein the moiety capable of specificbinding to FAP comprises (a) a VH domain comprising (i) CDR-H1comprising the amino acid sequence of SEQ ID NO:9, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO:10 and (iii) CDR-H3comprising the amino acid sequence of SEQ ID NO:11, and a VL domaincomprising (iv) CDR-L1 comprising the amino acid sequence of SEQ IDNO:12, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:13 and(vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO:14, or (b) aVH domain comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:104, (ii) CDR-H2 comprising the amino acid sequence of SEQ IDNO:105 and (iii) CDR-H3 comprising the amino acid sequence of SEQ IDNO:106, and a VL domain comprising (iv) CDR-L1 comprising the amino acidsequence of SEQ ID NO:107, (v) CDR-L2 comprising the amino acid sequenceof SEQ ID NO:108 and (vi) CDR-L3 comprising the amino acid sequence ofSEQ ID NO:109.
 19. The TNF family ligand trimer-containing antigenbinding molecule of any one of claims 1 to 18, wherein the moietycapable of specific binding to FAP comprises a variable heavy chaincomprising an amino acid sequence of SEQ ID NO:16 and a variable lightchain comprising an amino acid sequence of SEQ ID NO:17 or wherein themoiety capable of specific binding to FAP comprises a variable heavychain comprising an amino acid sequence of SEQ ID NO:110 and a variablelight chain comprising an amino acid sequence of SEQ ID NO:111.
 20. TheTNF family ligand trimer-containing antigen binding molecule of any oneof claims 1 to 16, wherein the target cell antigen is CEA.
 21. The TNFfamily ligand trimer-containing antigen binding molecule of any one ofclaim 1 to 16 or 20, wherein the moiety capable of specific binding toCEA comprises a VH domain comprising (i) CDR-H1 comprising the aminoacid sequence of SEQ ID NO:112, (ii) CDR-H2 comprising the amino acidsequence of SEQ ID NO:113 and (iii) CDR-H3 comprising the amino acidsequence of SEQ ID NO:114, and a VL domain comprising (iv) CDR-L1comprising the amino acid sequence of SEQ ID NO:115, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:116 and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:117.
 22. The TNF familyligand trimer-containing antigen binding molecule of any one of claim 1to 16 or 20 or 21, wherein the moiety capable of specific binding to CEAcomprises a variable heavy chain comprising an amino acid sequence ofSEQ ID NO:118 and a variable light chain comprising an amino acidsequence of SEQ ID NO:119.
 23. The TNF family ligand trimer-containingantigen binding molecule of any one of claims 1 to 16, wherein thetarget cell antigen is CD19.
 24. The TNF family ligand trimer-containingantigen binding molecule of any one of claim 1 to 16 or 23, wherein themoiety capable of specific binding to CD19 comprises (a) a VH domaincomprising (i) CDR-H1 comprising the amino acid sequence of SEQ IDNO:120, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:121and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:122,and a VL domain comprising (iv) CDR-L1 comprising the amino acidsequence of SEQ ID NO:123, (v) CDR-L2 comprising the amino acid sequenceof SEQ ID NO:124 and (vi) CDR-L3 comprising the amino acid sequence ofSEQ ID NO:125, or (b) a VH domain comprising (i) CDR-H1 comprising theamino acid sequence of SEQ ID NO:128, (ii) CDR-H2 comprising the aminoacid sequence of SEQ ID NO:129 and (iii) CDR-H3 comprising the aminoacid sequence of SEQ ID NO:130, and a VL domain comprising (iv) CDR-L1comprising the amino acid sequence of SEQ ID NO:131, (v) CDR-L2comprising the amino acid sequence of SEQ ID NO:132 and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO:133.
 25. The TNF familyligand trimer-containing antigen binding molecule of any one of claim 1to 16 or 23 or 24, wherein the moiety capable of specific binding toCD19 comprises a variable heavy chain comprising an amino acid sequenceof SEQ ID NO:126 and a variable light chain comprising an amino acidsequence of SEQ ID NO:127 or wherein the moiety capable of specificbinding to FAP comprises a variable heavy chain comprising an amino acidsequence of SEQ ID NO:134 and a variable light chain comprising an aminoacid sequence of SEQ ID NO:135.
 26. The TNF family ligandtrimer-containing antigen binding molecule of any one of claims 1 to 25,wherein the Fc domain is an IgG, particularly an IgG1 Fc domain or anIgG4 Fc domain.
 27. The TNF family ligand trimer-containing antigenbinding molecule of claim 26, wherein the Fc domain is an IgG1 Fc domaincomprising the amino acid substitutions at positions 234 and 235 (EUnumbering) and/or 329 (EU numbering).
 28. The TNF family ligandtrimer-containing antigen binding molecule of any one of claims 1 to 27,comprising one moiety capable of specific binding to a target cellantigen.
 29. The TNF family ligand trimer-containing antigen bindingmolecule of any one of claims 1 to 28, wherein the antigen bindingmolecule comprises (i) a first heavy chain comprising the VH domain of aFab molecule capable of specific binding to a target cell antigen, (ii)a light chain comprising the VL domain of a Fab molecule capable ofspecific binding to a target cell antigen, and (iii) a second heavychain comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:103or SEQ ID NO:8.
 30. The TNF family ligand trimer-containing antigenbinding molecule of any one of claims 1 to to 29, wherein the antigenbinding molecule comprises (i) a first heavy chain comprising a VHdomain comprising an amino acid sequence of SEQ ID NO:16 or a VH domaincomprising an amino acid sequence of SEQ ID NO:110, (ii) a light chaincomprising a VL domain comprising an amino acid sequence of SEQ ID NO:17or a VL domain comprising an amino acid sequence of SEQ ID NO:111, and(iii) a second heavy chain comprising the amino acid sequence of SEQ IDNO:5 or SEQ ID NO:103 or SEQ ID NO:8.
 31. The TNF family ligandtrimer-containing antigen binding molecule of any one of claims 1 to 29,wherein the antigen binding molecule comprises (i) a first heavy chaincomprising a VH domain comprising an amino acid sequence of SEQ IDNO:118, (ii) a light chain comprising a VL domain comprising an aminoacid sequence of SEQ ID NO:119, and (iii) a second heavy chaincomprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:103 orSEQ ID NO:8.
 32. The TNF family ligand trimer-containing antigen bindingmolecule of any one of claims 1 to 29, wherein the antigen bindingmolecule comprises (i) a first heavy chain comprising a VH domaincomprising an amino acid sequence of SEQ ID NO:126 or a VH domaincomprising an amino acid sequence of SEQ ID NO:134, (ii) a light chaincomprising a VL domain comprising an amino acid sequence of SEQ IDNO:127 or a VL domain comprising an amino acid sequence of SEQ IDNO:135, and (iii) a second heavy chain comprising the amino acidsequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8.
 33. The TNFfamily ligand trimer-containing antigen binding molecule of any one ofclaims 1 to 27, comprising two moieties capable of specific binding to atarget cell antigen.
 34. The TNF family ligand trimer-containing antigenbinding molecule of any one of claim 1 to 27 or 33, wherein the antigenbinding molecule comprises (i) a first heavy chain comprising a VHdomain of a Fab molecule capable of specific binding to a target cellantigen, (ii) two light chains comprising each the VL domain of a Fabmolecule capable of specific binding to a target cell antigen, and (iii)a second heavy chain comprising a VH domain of a Fab molecule capable ofspecific binding to a target cell antigen and the amino acid sequence ofSEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8.
 35. The TNF family ligandtrimer-containing antigen binding molecule of claim 34, wherein theantigen binding molecule comprises (i) a first heavy chain comprising aVH domain comprising an amino acid sequence of SEQ ID NO:16 or a VHdomain comprising an amino acid sequence of SEQ ID NO:110, (ii) twolight chains comprising each a VL domain comprising an amino acidsequence of SEQ ID NO:17 or a VL domain comprising an amino acidsequence of SEQ ID NO:111, and (iii) a second heavy chain comprising aVH domain comprising an amino acid sequence of SEQ ID NO:16 or a VHdomain comprising an amino acid sequence of SEQ ID NO:110 and the aminoacid sequence of SEQ ID NO:5 or SEQ ID NO:103 or SEQ ID NO:8.
 36. TheTNF family ligand trimer-containing antigen binding molecule of claim34, wherein the antigen binding molecule comprises (i) a first heavychain comprising a VH domain comprising an amino acid sequence of SEQ IDNO:118, (ii) two light chains comprising each a VL domain comprising anamino acid sequence of SEQ ID NO:119, and (iii) a second heavy chaincomprising a VH domain comprising an amino acid sequence of SEQ IDNO:118 and the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:103 orSEQ ID NO:8.
 37. The TNF family ligand trimer-containing antigen bindingmolecule of claim 34, wherein the antigen binding molecule comprises (i)a first heavy chain comprising a VH domain comprising an amino acidsequence of SEQ ID NO:126 or a VH domain comprising an amino acidsequence of SEQ ID NO:134, (ii) two light chains comprising a VL domaincomprising an amino acid sequence of SEQ ID NO:127 or a VL domaincomprising an amino acid sequence of SEQ ID NO:135, and (iii) a secondheavy chain comprising a VH domain comprising an amino acid sequence ofSEQ ID NO:126 or a VH domain comprising an amino acid sequence of SEQ IDNO:134 and the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:103 orSEQ ID NO:8.
 38. An isolated polynucleotide encoding the TNF familyligand trimer-containing antigen binding molecule of any one of claims 1to
 37. 39. A vector, particularly an expression vector, comprising theisolated polynucleotide of claim
 38. 40. A host cell comprising theisolated polynucleotide of claim 38 or the vector of claim
 39. 41. Amethod for producing the TNF family ligand trimer-containing antigenbinding molecule of any one of claims 1 to 37, comprising the steps of(i) culturing the host cell of claim 40 under conditions suitable forexpression of the antigen binding molecule, and (ii) recovering theantigen binding molecule.
 42. A pharmaceutical composition comprisingthe TNF family ligand trimer-containing antigen binding molecule of anyone of claims 1 to 37 and at least one pharmaceutically acceptableexcipient.
 43. The TNF family ligand trimer-containing antigen bindingmolecule of any one of claims 1 to 37, or the pharmaceutical compositionof claim 38, for use as a medicament.
 44. The TNF family ligandtrimer-containing antigen binding molecule of any one of claims 1 to 37,or the pharmaceutical composition of claim 42, for use in the treatmentof cancer.
 45. Use of the TNF family ligand trimer-containing antigenbinding molecule of any one of claims 1 to 37 for the manufacture of amedicament for the treatment of cancer.
 46. A method of treating adisease in an individual, comprising administering to said individual atherapeutically effective amount of a composition comprising a TNFfamily ligand trimer-containing antigen binding molecule of any one ofclaims 1 to 37 in a pharmaceutically acceptable form.
 47. The method ofclaim 46, wherein said disease is cancer.